CN101065502B - Microarray-mediated diagnosis of herpes virus infection by monitoring host's differential gene expression upon infection - Google Patents

Abstract

The present invention discloses molecule and test about diseases can be used for diagnosing and evaluating the animals infected by herpes virus, and for testing the animals in herpes virus infection or in sequela. The invention allows early diagnosis and monitoring of an infected animal's immune response and thus enables better treatment and management decisions to be made in clinical and sub-clinically affected animals.

Description

Carry out microarray-mediated herpesvirus infection diagnosis through detecting the distinctiveness genetic expression of infecting the back host

Invention field

Present invention relates in general to diagnosis, detect host response, the method and system of herpesvirus infection in monitoring, treatment and the processing Mammals.The present invention can be applied to early diagnosis and infect, detects the specific immune response (having or do not have clinical symptom) to herpesvirus infection, can be to receiving the clinical and subclinical Mammals that influences to make better treatment and handling decision.The present invention also can be applied to monitor the Mammals of the clinical sign recurrence that the relevant sequela risk of bleb takes place or cause because of latent virus.This Mammals includes but not limited to: because of suffer from other disease or give due to the medicine, suffer from chronic fatigue syndrome, meet with stress or the animal that weakens of training immunity.

Background of invention

Simplexvirus has been represented and has extensively caused people and the domestic and viral extended familys wildlife various diseases.Known herpes virus type surpasses 80 kinds, but has only 8 kinds knownly can cause the human disease.These 8 kinds are divided into three subfamilies shown in the following table.

Herpesvirus infection distributes extensively, and the disease that causes has serious Economic Importance.In fact, the Mammals of most of kinds is in the early stage at least a virus strain infection that once received simplexvirus of life.Infection is persistent, and does not have known treat-ment.Can use antiviral to treat, but after symptom takes place, use and/or take (when they have the time spent at least) often, because spinoff is with expense and can not be as preventing administration.Therefore, their application is generally limited to the mankind.

At present can only detect clinical sign and the antibody that back and patient produce behind other zoogenetic infection occur based on the diagnostic method of antibody test.Other detection method comprises PCR amplification virus transcription thing or viral DNA, tissue culture isolated viral and/or virus neutralization tests.

All herpesvirus infections are persistent.After the primary infection, virus gets into latent period.When patient's immunizing power weaken or receive stress or strong movements when training, disease can activate again.Again the activation of virus can cause symptomatic recurrence, or general malaise, or the tolerance or the performance of taking exercise reduced, perhaps can be asymptomatic.

The mensuration of antiviral antibody, antigen or transcript (value) not necessarily is associated with disease or clinical sign.

The available effective vaccine does not almost have.

But medicament is treated, but their costlinesses need give before main clinical sign occurs for obtaining greatest treatment efficacy.

Epidemiology and clinical manifestation

Simplexvirus can cause several kinds of clinical manifestations in the host that normal and immunizing power weaken.Asymptomatic during most of herpesvirus infection, most of cases that bleb is propagated are people that outburst fashion is not recognized or still do not diagnosed out.

The adult of 50-90% has the antibody to HHV-1 or 1 type human herpes simplex vicus (HSV-1); The 20-30% adult has the antibody to HHV-2 or 2 type human herpes simplex vicuses (HSV-2).(virus) propagation in the low colony of socio-economic status chooses friends indiscriminately individuality with property is more general.HSV-1 is usually relevant with actinal surface portion (orofacial) regional primary infection, the latent infection gasserian ganglion, and HSV-2 is common relevant with genital infection, latent infection rumpbone neuroganglion.Though initial and recurrent infection is (self-limited) of self-limit normally, HSV can cause serious disease, newborn infant's dissemination bleb for example, viral encephalitis and blinding property keratitis (blinding keratitis).

During acute primary infection, HSV hide lastingly with skin that gets into or the corresponding nerve root ganglion of mucosal sites (nerve root ganglia) in.In lip (orolabial) infected, HSV hid in gasserian ganglion, and when sexual organ or anorectal infection, hides in the rumpbone neuroganglion.Various stimulations, for example the damage of UV-light and sensory nerve can activate the HSV that hides again.The recurrence venereal disease is decreased and to be occurred in or near the primary infection position.Recurrence as if with some mode reduce individual relevant to the factor of disease resistance, for example flu or upper respiratory tract infection, the HS physical sport, Exposure to Sunlight, stress, the woman month warp triggers because of food with some individualities, particularly a large amount of chocolates or peanut.

During reactivation, HSV duplicates in neuroganglion, and the progeny virus particle moves to the mucous membrane or the epithelial surface of its reactivation neuroganglion domination to outer planet along sensory nerve.Virus is in the active typical clinical sign and sick the damage (situation that recurs flu as the people who infected by HSV is the same) that has produced the HSV infection and recurrence of duplicating of skin surface then.

" Epstein-Barr virus " (EBV) or HHV-4 and the infectious mononucleosis and the tumour that are also referred to as " glandular fever " to form (for example, Burkitt lymphoma and nasopharyngeal carcinoma (nasopharyngeocarcinoma)) relevant.In addition, in the patient of immunosuppression and trouble Hodgkin, find to have EBV.EBV has in the whole world, and most of people were infected by it once in life at it.In the U.S., nearly 95% adult is infected in 35-40 when year.In case maternal antibody provide protection (existing during birth) disappears, the baby is promptly to the EBV susceptible.

The EBV symptom of infectious mononucleosis comprises heating, throat pain, lymph gland swelling.Spleen or liver swelling can take place sometimes.Rare cardiac problems or relate to cns, infectious mononucleosis is fatal scarcely ever.The most of individualities that contact with the people who suffers from infectious mononucleosis were infected by EBV in the past, did not have the risk of suffering from infectious mononucleosis.In addition, the EBV propagation need contact with sufferer's saliva (in mouth, finding virus) closely.This virus is not generally through air or blood propagation.Latent period, or from infect to the time that symptom occurs be 4-6 week.The infectious mononucleosis patient propagates this infection to other people in can be in several weeks.Yet, do not recommend special preventive measures or partition method, because in the saliva of healthy subjects, often can find this virus yet.In fact, many healthy persons can carry and intermittently propagate this virus all the life.These people are the main source (primary reservoir) of person to person's propagation normally.In view of this, possibly prevent this virus disseminating hardly.

Though the symptom of infectious mononucleosis can disappear 1 or 2 middle of the month usually, Epstein-Barr virus can be kept in the few cell that lies dormant or latent in throat and blood in all the other times in this person's all one's life.This virus can periodically activate again, is common in sufferer's the saliva.This reactivation does not often have obvious disease symptoms when taking place, but nonspecific symptom can be arranged, and for example discomfort or athletic performance are not good.EBV also can set up lifelong dormancy and infect in some cells of body immune system.Two kinds of rare cancers of seldom finding in the U.S. Burkitt lymphoma and nasopharyngeal carcinoma can appear, in this virus few carrier afterwards.As if EBV plays an important role in these malignant tumours take place, but possibly not be the sole cause of these diseases.

Cytomegalovirus (CMV) or HHV-5 generally are shown in all geographic areas and the colony of social economy, and in the U.S., 40 years old adult of 50%-85% is infected.CMV can cause immunosuppression person's pulmonary infection.In addition, it is believed that CMV is relevant with EBV and chronic fatigue syndrome, per 100,000 philtrums have 6 people influenced by this disease.Can discharge infectious CMV in the infected's the body fluid before any, thereby can in urine, saliva, blood, tears, seminal fluid and breast milk, find.The discharge of virus can intermittently take place, and does not have detectable sign, does not also cause symptom.CMC is in interpersonal propagation.CMV can also can propagate through breast milk, transplanted organ through spreading through sex intercourse, and seldom propagates through blood transfusion.Though should virus not be hyperinfection property, its demonstration can be propagated within the family with among the young children.Often can stop the propagation of this virus,, should virus be absorbed then by susceptible person's nose or mouth because its modal circulation way is to contact with hand through infected body fluid.Therefore, should the heart when the article of handling children such as diaper.With soap and water simply wash one's hands can be effectively with should virus from removing on hand.

The symptom that CMV causes is less than EBV, and long-term unconspicuous infection often takes place, and virus resides in the cell and do not cause detectable damage or clinical disease during this period.Because of taking medicine or disease causes body immune system to be badly damaged making often this virus from hiding or dormant state activates again.Cmv infection is common but do not have symptom in baby and child; Therefore, known infected children are dropped out of school or drop out of school unreasonable also unnecessary.Similarly, inpatient does not need independent or meticulous isolation preventive measures.The value of children or patient being carried out the CMV screening is suspicious.The cost of this method is unrealistic with operation.Should the known children that infected CMV not got rid of separately, isolate or treat especially.On the contrary, suggestion is educated the personnel that take care of children and is set up effective hygiene measure.It is gestation that CMV possibly cause the situation of problem, the people of baby and child care work and the people that immunizing power weakens.

Varicella zoster virus (VZV) or HHV-3 cause children varicella usually, and its (infection) needs suction the infected's nose or throat to drain into the small droplets that contains virion of air.This virus gets into human body through the cell that infects respiratory tract, thereby diffuses to other position of great majority of health, comprises skin and causes the characteristic fash.Each sick decrease (spot) has experienced the series of features sexual stage in an about week.Papule and vesicle develop into warts, incrustation then, healing.The notable feature of varicella is to grow several consecutive spots, reaches the peak period (occur first fash 3-4 days after) of disease, develops into all ill damage of all stages of incrustation from new bubble.

The ability that VZV propagates in this way shows that varicella has infectivity.Virus is discharged from air flue and is started from the later stage of hiding, and continues to institute's spottiness incrustation.Though skin blister contains virion, they are not main contagium.Scar does not have infectivity.The interference method that is of long duration is to reduce heating and uncomfortable (that is, antipyretic, cooling bath and comfort property washing lotion (soothing lotion)) as far as possible.

Varicella is the clinical manifestation of VZV primary infection.After the primary infection recovery, VZV does not disappear in body, but sensorineural root is in dormant state (but common many decades) in spinal cord.After infecting activation again, cause infected sensory nerve institute's distributed areas pain and fash.Latent infection can cause the zoster outbreak.This takes place in the elderly usually, possibly be that immunity system can not be controlled should virus because with age.The fash of zoster contains the VZV particle, as the fash of varicella.Therefore, zoster infects varicella to the people's who does not suffer from the past varicella risk little.The baby usually can be through catching varicella with grand parents's intimate contact of suffering from zoster, but this propagation risk is low, because VZV does not discharge through throat during the zoster.

Roseola virus or HHV-6 infect relevant with " roserash " and " baby's rash (infantum) " that children and immunizing power weaken the patient.For example, AIDS patient shows HHV-6 and infects, though the meaning that HHV-6 infects it be unclear that.HHV-6 enantiopathy cytotoxic drug is responsive.Yet how antiviral is resisted HHV-6 or should virus how the resistance of this medicine to be produced it be unclear that.The meaningful aspect of HHV-6 is that it infers that it is relevant with multiple sclerosis and chronic fatigue syndrome respectively.

HHV-7 and HHV-8 (elongated virus) are understood still less.The clear and definite evidence that HHV-7 participates in any human diseases directly also is not reported.Yet research shows that HHV-7 maybe be relevant with the HHV-6 infections relating.One relevant viewpoint is that it is relevant with Kaposi sarcoma to it is believed that HHV-8 infects.

In addition, think that simplexvirus is the reason of motor capacity of major reason and the heavy damage horse of the loss of horse keeping industry.Animal doctor, trainer and horse owner people tend to only rely on the horse that experiential therapy is suffered from respiratory tract disease, in default of clinical guidelines and laboratory method, also because do not understand virus and Secondary cases infectation of bacteria and the relation of disease between the time length.Other diagnosis or appraisal procedure complicacy, invasive, inconvenience, costliness, consuming time, animal often possibly and often need be transported to diagnositc center with animal because of injured dangerous of this method.

In the research that carry out the West Australia, have the blood of the horse that respiratory problems or (motion) perform poor from 48% and to be separated to simplexvirus.Yet, also can from 54% do not have clinical sign Malaysia and China be separated to simplexvirus, thereby reach a conclusion: having this virus in the hemocyte is not the determinative of disease.Isolated viral more can show respiratory tract infection from the nose swab, but only in 50% clinical case, is separated to.According to reports Britain nearly 75% horse carry this virus.

Equine rhinopneumonia is generally acknowledged equine disease different by two kinds but that antigenicity associated virus (being called 4 type EHVs (EHV-4) and 1 type EHV (EHV-1)) causes with equine abortion.EHV-1 is the cause of disease of the popular miscarriage of horse, perinatal death, respiratory tract disease and neurosigns (once in a while).Miscarriage is the surprising and the most fearful result that EHV infects, and it is raiser's economic woes, causes customer churn and a large amount of insurance benefits.The respiratory tract disease that EHV-1 or extremely relevant EHV-4 cause can poorly influence the sports performance of horse.

Show that in the research that Australian Hunter Valley carries out the EHV-1 infection is popular in the horse keeping industry, young coltfoal is infected by EHV-1 often before these place 60 ages in days.The independent studies of carrying out in the U.S. shows young coltfoal 6-8 month seropositive conversion after wean of 85%.It is believed that young coltfoal is to contact through the respiratory droplets with mare or the young coltfoal of same crowd to infect.

EHV-1 is a kind of DNA α simplexvirus of having a preference for airway epithelial cell.This virus is transmitted to other organ through immune system cell at whole body.Because these two kinds of viruses are relevant on antigenicity, can't confirm whether horse receives among EHV-4 or the EHV-1 one or both infection through serologic test (blood test) at present.For example, if infected by EHV-4 as the horse of young coltfoal, the antibody that produces in its serum can not only also can react with EHV-1 with EHV-4, therefore, can't know whether this young coltfoal infected by EHV-4.EHV-4 only confirms to cause respiratory tract disease; And EHV-1 can also cause neural and reproductive disease (Wilcox and Raidal; 2000; " effect of virus in respiratory tract disease " (Role of viruses inrespiratory disease), RIRDC publication number 00/146, RIRDC item number UMU-22A; Dunowska etc., 2002, New Zealand Veterinary Journal50 (4): 132-139; Dunowska etc., 2002, New Zealand Veterinary Journal50 (4): 140-147).

Proved that EHV-1 is lasting, lifelong latent infection, it activates again and causes the horse respiratory tract disease to be shown effect once more.Yet when other horses that infect when contacting with first dry goods (index case (index case)) were the pregnant mare in the pasture, this virus activated again and it is propagated to other pregnant mare that contacts with it, and its consequence is more serious to raising the farm.One or many abortion of mares that contact with it possibly miscarried or cause to index case mare oneself.Fetus, fetal membrane and the liquid of miscarriage receive the EHV-1 severe infections, have polluted the place that miscarriage takes place.Born other curious mare comes the miscarriage place in this pasture, smells this fetus and peptide film.Like this, in this pasture almost 100% mare infected miscarriage in 10 or 20 days, thereby " the miscarriage storm " that cause being commonly referred to.This outburst of EHV-1 miscarriage is to global horse keeping industry, and particularly thoroughbred (Thoroughbred) and standard-bred (Standardbred) have Economic Importance.

Immunizing power and diagnosis

Simplexvirus can be induced strong humoral antibody reaction, though this reaction protection host's validity is doubtful.Finally be through cell-mediated mechanism, for example cytotoxic lymphocyte or natural killer cell are given provide protection.One of key feature of herpesvirus infection is lifelong long-lasting infection and hides.This virus maintains in the nucleus, after clinical symptom disappears for a long time still can from many organ separation to.Therefore, when patient (horse) has the clinical sign that is difficult to describe, for example (motion) perform poor or be uncomfortable, can be from the separate tissue to virus, it be unclear that but whether the activation of virus is the reason of these symptoms.Stress or other factors can cause dormancy activated viral and the clinical sign followed (Walker etc., 1999, Veterinary Microbiology, 68:3-13).

HSV-1 or HSV-2 infect the immunizing power of ability inducing cell mediation, produce the antibody of general type and particular type.Do not influence generation or the recurrence frequency that HSV hides though these immune mechanisms it seems, activate again in case (virus) takes place, the seriousness of their adjustable clinical recurrences also reduces HSV and duplicates.

HSV-1 or HSV-2 infect the host immune response that causes and show to provide and resist the partial protection effect that HSV subsequently infects because in the individuality that HSV infects common resistibility to self infection.In addition, with the physiognomy ratio that did not infect in the past HSV, when the patient who infected HSV was infected by another HSV type, clinical sign often more relaxed.

HSV is the most frequent detected virus in the diagnostic test chamber.Can be through virus separation, polymerase chain reaction (PCR) (Espy etc., 2000, J Clin Microbiol., 38 (2): 795-799) make diagnosis with histopathology (method).These methods all can not be used for control and monitoring of diseases.Other laboratory inspection that can be used for diagnosing comprises scrapes bits (scrapings) and blood antibody test with the microscopy special disposal.Some detect only effective in early days, possibly need multiple these checks for confirming that bleb exists.Genital herpes may mistaken diagnosis be other disease, comprises syphilis.Serum antibody level height also is the index of recent infection.If someone has visible symptom really, cultivate check in preceding 48 hours after being recommended in symptom and occurring.After 48 hours, the risk that obtains the false negative assay is arranged because symptom possibly begin to fully recover and skin on do not have enough virus to cultivate.

When someone does not have visible symptom, but when worrying herpes infection is arranged, can adopt blood test.In fact blood test is not to detect virus; But look for antibody (immunoreation of body), but the human blood of 50-90% has positive antibody.Available twice blood test at present obtains the precise results of bleb.The same with any blood test, it is oral cavity or reproductive tract that these checks can not be measured infection site.Yet because the most of cases of genital herpes are that HSV-2 causes, 2 type antibody results are positive, and most probable shows it is genital herpes.With regard to the most accurate result, recommend from waiting until that 12-16 has the enough time to produce antibody after week at least possible last duration of contact with bleb (virus).

According to the clinical diagnosis of generating heat, having sore throat, lymph gland swelling symptom and patient's age proposing EBV and infectious mononucleosis.Usually need laboratory inspection to confirm.Infectious mononucleosis patient's serology result comprises that white blood cell count(WBC) raises, some atypia white corpuscle per-cent raises and " single spot " check is positive.

Through enzyme-linked immunosorbent assay (or ELISA), a kind of serologic test that detects antibody carries out the CMV clinical diagnosis.Can utilize its result to confirm whether the baby has the maternal antibody of acute infection, infection in the past or passive acquisition.Other check comprises various fluorescent tests, IHAT and milk agglutination test.

According to respiratory symptom, promptly cough or nasal mucus carry out the EHV diagnosis.Yet, distinguish respiratory bacillary infection or viral infection, exercise inductive lung is hemorrhage with irritated most important.The cost of mistaken diagnosis is very big.The cost of horse owner's diagnose medical conditions comprises transportation, animal doctor's expense and pathological examination.Respiratory tract disease can kill (horses) fast, produces lifelong deformity, hinders (motion) performance or need long-term rest.Be in the horses that challenge virus carries situation and can infect other animal again.

With horse keeping already is example, need whether have that EHV-4 and/or EHV-1 antibody carry out accurately to horses, the monitoring of the serology of type specific helps the epidemiology (characteristics) that we understand these virus, particularly EHV-1.EHV-1 infects and is difficult to diagnosis and treatment, any useful information how the sector welcome treats ill horses.Yet, can not distinguish EHV-1 or EHV-4 antibody in the polyclonal serum at present, because between these two kinds of viruses antigenicity cross reaction is widely arranged.Adopt this specific serum to learn check control (perhaps eradicating) EHV-1 is also had far reaching significance with the candidate horses of selecting vaccine inoculation.Receive short duration protection opposing EHV-1 though infect the back horses, do not have enough high-caliber permanent immunity power to come to resist consistently the EHV-1 disease usually.Therefore, but horses its in life repeated infection for several times, and simplexvirus can be set up lifelong latent infection and made the vaccine inoculation scheme complicated in host animal.

Still there is not to prevent the vaccine of HSV disease generation.Though the some protein subunit vaccine that is the basis with the HSV-2 envelope glycoprotein and then the back clinical trial phase.Select these antigens to be because they are the target and the ability trigger cell immunizing power of neutralizing antibody reaction.

Developed oral antiviral, for example acyclovir, Famciclovir (famcyclovir) or valacyclovir (valacyclovir) come effectively treatment bleb (virus) infection.These medicines can be used for treating the bleb outburst or can be used for suppressing the bleb recurrence.Lower dosage helps to reduce the number of times of bleb outbreak among the frequent outburst crowd.Ganciclovir, penciclovir (penciclovir) and acyclovir are effective suppressor factor of 1 type (HSV-1) and 2 types (HSV-2) hsv.This antiviral therapy is expensive, need when clinical sign outbreak the earliest, give.These antiviral therapies are based on the employing suicide gene, for example the thymidine kinase gene.Having compared ganciclovir, penciclovir and acyclovir induces hsv thymidine kinase (HSVTK) system to cause effect (Shaw etc., 2001, Antivir Chem Chemother., 12 (3): 175-86) of necrocytosis.All compound retards the HSVTK transformant growth or reduced its survival.

Except treating these symptoms, EBV and infectious mononucleosis there is not special treat-ment.There are not antiviral or vaccine to use.Some physicians have opened 5 days steroid and have controlled throat and tonsilla swelling the course of treatment.Have report to say that the use sterol can reduce the total time and the seriousness of disease, but these reports are not delivered.At last, even detect EBV antibody, when for example EA test prompting existed reactivation to infect, this result showed that not necessarily the present medical conditions of patient is due to EBV infects.Many healthy persons that do not have a symptom can have the antibody of EBV EA for many years after infecting EBV first.

At present, the cmv infection that exists in the healthy individuals needn't be treated.Existing just assessment antiviral therapy in the baby.Ganciclovir is used to treat immunosuppression and the patient who suffers from visual correlation or life-threatening disease.Vaccine still is in the research and development stage.

Usually antiviral compound treatment varicella that need not be special is because its time length is short and common gentle character is uncomplicated.Some doctors believe that antiviral is suitable for the elderly that disease maybe be more serious.Chickenpox vaccine (varicella virus (varicella) vaccine) was brought into use from nineteen ninety-five.Research shows that the prophylactic validity of Varivax is 85%.This vaccine is of value to the grownup of no immunizing power, particularly is in high risk grownup, for example child care and health worker.Because most of grownups have immunizing power, be recommended in the preceding inspection of vaccine inoculation serology state.

The main difficult point of treatment zoster is a fast pain relief.Four kinds of factors have increased the risk of lasting pain independently: age growth, the pain (being called forerunner's pain) when fash occurs before serious or medium serious pain (being called acute pain), the fash appearance occur failing to obtain enough antiviral therapies in 3 days with fash.Think pain, due to the infected nerve injury that particularly lasting pain is virus induction to a great extent.The principle of using antiviral is very simple: the virus replication that stops through fast as far as possible reduces as far as possible nerve injury.Zoster is to oral antiviral, and promptly acyclovir, Famciclovir and valacyclovir truly have reaction.

The early stage EHV-1 of evaluation infects, and particularly virus abortion is most important for the treatment horses, thereby makes the susceptible horses can repeated infection of generating period property and recurrence.Because cause training fate forfeiture, repeated infection and palindromia, miscarriage and (motion) to perform poor, the cost that the EHV infection already causes horse keeping is very big.Treatment mainly depends on accurate diagnosis.Vaccine capable of using causes strong humoral immune reaction, but these vaccines complete protection of tool not.Sudden infection (breakthrough infection) betides the vaccine inoculation animal usually.Though can adopt vaccine, generally know that the provide protection that they provide is very few, and sudden infection often takes place in the vaccine inoculation animal.Antybody therapy can only prevent the Secondary cases infectation of bacteria.

At present, the simplexvirus in the diagnosis blood and the method for relative disease are according to antibody-antigen quantitatively determined or detect viral genetic information (for example, through the polymerase chain reaction).For example, USP 6,506,553 have described the test of diagnosing EBV and relative disease through the antigen-antibody in the detection blood sample.This test detects in the blood, more particularly the IgG and the IgM antibody of the dispersivity of EBV EA (EA-D) and restricted (EA-R) component in the serum.Can adopt this to test and diagnose the EBV-relative disease, for example infectious mononucleosis (IM); Also can be used for distinguishing the acute phase and the convalescent individuality of this disease.Yet the method that this patent is described is to detect EA-Ab, rather than detects virus or to this viral immunoreation.

USP 6,537,555 have described the compsn and the method for diagnosing and treating the HSV infection according to HSV detection of antigens (result).Yet this patent is not described and is detected virus or to the immunoreactive method of this virus.

Genetic expression that international publication WO99/45155 describes and molecular diagnosis method are used for amplification and detect EBV nucleic acid, particularly RNA specific sequence.This method is specially adapted to detect in the circulation PBC, (utilizing " in the solution " amplification or " original position " amplification technique) in people's (tumour) tissue sample and the thin section thereof, with other biological sample that possibly contain the EBV cells infected in late period of EBV genetic expression infect.Yet this method only detects the virus transcription thing, is suitable for the terminal illness diagnosis most.This method does not detect causing the immunoreations of the virus infection of symptom the earliest such as discomfort, heating and swollen lymph node yet.

U.S. Patent Application Publication 20040072147 discloses utilizes probe oligonucleotides and at least two kinds of primer tasteless nucleotides to come the target area section of following particular type simplexvirus of selective amplification or strain to comprise: HSV-1, resistance HSV-1, HSV-2, resistance HSV-2, VZV, EBV (HHV-4a and HHV-4b), CMV, lymph cryptovirus (HHV-6a, HHV-6b), HHV-7 and elongated virus (HHV-8).Yet this method can not provide when infected whether disease stage or animal or disease be any information of active.

USP 6,193,983 have described the method that detects EVH-4 and EVH-1 type specificity gp is used for relevant with the characteristic of this gp clinically field.This method detects anti-EHV-1 and EHV-4 specific antibody, but it is contacted in childhood and these virus to know most of horses, the sustainable certain hour of antibody titers.So this method can not provide when infected whether disease stage or animal or disease be any information of active.

In a word, the various virus strain infections of simplexvirus are general phenomenon in the community, cause having the disease of serious economic impact.Most of people and domestic animal are in the infection that all receives at least a strain of simplexvirus in petticoats, and this infection is lifelong.Simplexvirus gets into latent period, can activate again and causes symptomatic recurrence.The reactivation frequentation of virus is toward asymptomatic, but can show as discomfort or nonspecific symptom, for example low fever, drowsiness, confirmed fatigue, exercise tolerance is not good or athletic performance is not good.Physiological stress (for example, heavy exercise, concurrent disease, stress), or immunity system weakens (for example HIV infect, immunosuppressant therapy) and can cause the simplexvirus reactivation and produce the chronic infection symptom.In view of these reasons, the monitoring herpesvirus infection is often important, particularly the patient that weakens of immunizing power or outstanding sportsmen.Present antibody diagnosis method itself is not monitored herpesvirus infection because the serum antibody that these methods detect 7-14 talent after infection raises and due to illness poison hide and continue and raise.Other present diagnostic method, for example virus is separated and PCR itself does not monitor (virus infection), because these method efforts or viral genome are not constantly to be present in the blood cell.The result that the current diagnosis method obtains does not join with the time correlation that clinical sign shows effect.For example, but at first can detect antibody and long-term existence in 10-14 days in the initial back of infecting.Depend merely on antibody test and can not show time or the disease activity level that takes place that infect, detect virus transcription thing or viral protein and also do not show the disease activity level.Host's immunity system finally is responsible for the provide protection that opposing virus is invaded.Be immunoreation, but rather than the viral clinical sign that causes this disease itself.The monitoring method that more is appropriate to herpesvirus infection is to detect the specific immune response of host to infecting.

Equally, need more efficient methods to diagnose herpesvirus infection at present, be fit to animal with antiviral treatment property or preventive therapy through host immune response mensuration activity herpesvirus infection and evaluation.In all cases, primary infection causes latent infection, and the patient that immunizing power weakens also often has risk of relapse.In this case, symptom maybe be obvious or not obvious, can detect maybe and can propagate.Therefore, need better method and reagent to assess and monitor the Mammals that is in herpesvirus infection and/or the risk of relapse at present.

The invention summary

The invention discloses the detection herpesvirus infection, particularly the method and system of reactivity herpesvirus infection.Identify and the gene of having described one group of predictability of the immune system cell that is used for (detection) herpesvirus infection.These genes and product thereof can be used for gene expression test, protein expression test, full test cell line and design and preparation therapeutical agent.They also can be used for measuring the animal that has or do not have the disease clinical sign.Someone proposes, and is frequent when opposing viral active indicator reaction when these tests, can obtain better that reply determines and regimen, comprises that outstanding sportsmen or immunizing power weaken the used scheme of patient.

The present invention has represented the marked improvement of present processing infected animals treatment technology.In some preferred embodiment, depend on the level that detects some mark, particularly circulating leukocyte in the host cell, rather than detect viral product or antiviral antibody.Equally, these methods are applicable to that extensive screening has symptom and asymptomatic animal.In some embodiment that with the circulating leukocyte is analytic target, can before detecting serum simplexvirus specific antibody, should detect the host at the very commitment of this virus progress, the particularly reaction of EHV infection to herpesvirus infection.

Therefore, the present invention is through detecting the host solves the diagnosis herpesvirus infection to the reaction of simplexvirus problem.Embodiment preferred comprises some expression of gene in the immune peripheral leukocytes of monitoring, and said genetic expression is reflected in the change that exists relevant rna level of herpesvirus infection or protein to produce pattern.

Therefore, on the one hand, the present invention provides the diagnostic test object, particularly has herpesvirus infection in the horses tested object, particularly the method for movable herpesvirus infection.These methods generally comprise and detect at least a unconventionality expression that is selected from following gene (this paper is also referred to as " herpesvirus infection marker gene " or " HVI marker gene ") in this tested object: (a) a kind of gene; Its polynucleotide expression product contain and below arbitrary sequence have at least 50% (and at least 51%-at least 99% and between all integer per-cents) nucleotide sequence of sequence homogeny: SEQ ID NO:1,2,4,6,7,8,10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113 (referring to table 1), or its complementary sequence; (b) a kind of gene, its polynucleotide expression product contain the nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114 (referring to table 1); (c) a kind of gene; Its polynucleotide expression product contain coding and below at least a portion of arbitrary sequence have at least 50% (and at least 51%-at least 99% and between all integer per-cents) nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) a kind of gene, its polynucleotide expression product contain at least can be under low preciseness, middle preciseness or high preciseness condition and the sequence of (a) and (b), (c) or the nucleotide sequence of its complementary sequence hybridization.These HVI marker gene of the present invention are at herpesvirus infection or suffer from abnormal expression in the animal of herpesvirus infection associated conditions, the exemplary of said illness comprise immunosuppression, stress, high-intensity athletic training, an accompanying infection and a special venereal disease disease.

This paper is called the polynucleotide expression product of used HVI marker gene " herpesvirus infection mark polynucleotide " or " HVI mark polynucleotide ".This paper is called the expression of polypeptides of HVI marker gene " herpesvirus infection mark " or " HVI labeling polypeptide ".

Therefore; In some embodiments; Said method comprises and detects the unconventionality expression be selected from following HVI mark polynucleotide: (a) contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of sequence homogeny nucleotide sequence: SEQ IDNO:1,2,4,6,7,8,10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114; (c) contain coding and below at least a portion of arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In other embodiments, said method comprises and detects the unconventionality expression that is selected from following HVI labeling polypeptide: (i) contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polypeptide of aminoacid sequence of sequence similarity: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114; The polypeptide that (ii) contains a following arbitrary sequence part: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, wherein said part contains at least 5 contiguous amino acid residues of this sequence; At least 15 contiguous amino acid residues of (iii) amino acid contained sequence and following arbitrary sequence have the polypeptide of 30% similarity at least: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114; The polypeptide that (iv) contains the part of following arbitrary sequence: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114; Wherein said part contains at least 5 contiguous amino acid residues of this sequence, and with immune interactional antigen binding molecules to take place with (i), (ii) or (iii) said certain sequence immunity takes place interact.

This unconventionality expression generally detects through following steps: the level or the functionally active of at least a HVI marker gene expression product in the biological sample that derives from tested object detected in (1); (2) with the level or the functionally active of detected each expression product and derive from one or multidigit normal subjects or one or multidigit (are not for example suffered from this disease; The level or the functionally active of corresponding expression product are made comparisons in the reference sample of object no activity infection); Wherein compare with the level or the functionally active of corresponding expression product in the reference sample; The level of this expression product or functionally active are variant in this biological sample, show to have herpesvirus infection or associated conditions in this tested object.In some embodiments; When this expression product or the detected level of each expression product or functionally active and corresponding this expression product or the detected level of each expression product or functionally active not simultaneously, said method also comprises existence, stage or degree or the associated conditions of herpesvirus infection in this tested object of diagnosis.In these embodiments; Compare with the level or the functionally active of each corresponding expression product; It is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% that the level of said each expression product of difference ordinary representation or functionally active improve at least, perhaps even at least improves about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%; Perhaps reduce about 10%, 20%, 30%40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99% at least; Perhaps even at least reduce about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, i.e. alleged " unconventionality expression " of hereinafter.In the exemplary of this type; Reduce existence or the associated conditions of measuring herpesvirus infection through detecting at least a level or the functionally active that is selected from following HVI mark polynucleotide: (a) contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQID NO:6,10,19,24,25,29,33,34,35,37,38,41,53,57,61,63,65,66,73,77,83,89,93,94,96,100,101,102,104,106,107 or 108, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:11,20,30,36,42,54,58,62,64,74,78,90,95,97,103 or 105; (c) contain coding and at least a portion of following sequence and have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:11,20,30,36,42,54,58,62,64,74,78,90,95,97,103 or 105, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In other exemplary; Confirm the existence or the associated conditions of herpesvirus infection through the raising that detects at least a level that is selected from following HVI mark polynucleotide or functionally active: (a) contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQ ID NO:1,2,4,8,12,13,15,17,21,23,26,27,31,39,43,45,47,49,51,55,59,67,69,71,75,76,79,81,85,87,91,98,99109,111 or 113, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:3,5,9,14,16,18,22,28,32,40,44,46,48,50,52,56,60,68,70,72,80,82,86,88,92,110,112 or 114; (c) contain coding and below arbitrary sequence at least a portion have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:3,5,9,14,16,18,22,28,32,40,44,46,48,50,52,56,60,68,70,72,80,82,86,88,92,110,112 or 114, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In some embodiments; When institute's detection level of this expression product or the detected level of each expression product or functionally active and corresponding this expression product or each expression product or functionally active were same or similar, said method comprised that also diagnosis does not exist herpesvirus infection or associated conditions.In these embodiments; The difference of detected level of the corresponding expression product with each of detected level of each expression product or functionally active or functionally active is no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or at 0.1% o'clock, hereinafter referred to as " normal expression ".

In some embodiments, said method comprises that detection is at least about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77 or the level or the functionally active of 78 kind of each expression product of HVI mark polynucleotide.For example, said method can comprise the level or the functionally active of independent detection a kind of HVI mark polynucleotide or joint-detection as many as 77,76,75,74,73,72,71,70,69,68,67,66,65,64,63,62,61,60,59,58,57,56,55,54,53,52,51,50,49,48,47,46,45,44,43,42,41,40,39,38,37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2 or a kind of other HVI mark polynucleotide.In another embodiment, said method can comprise the level or the functionally active of a kind of HVI labeling polypeptide of independent detection or joint-detection as many as 77,76,75,74,73,72,71,70,69,68,67,66,65,64,63,62,61,60,59,58,57,56,55,54,53,52,51,50,49,48,47,46,45,44,43,42,41,40,39,38,37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2 or a kind of other HVI labeling polypeptide.In the exemplary of this type; Said method comprises level or the functionally active that detects with at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16 or each expression product of 17 kind of HVI marker gene (hereinafter referred to as " the relevant HVI marker gene of one-level ") that have herpesvirus infection or associated conditions or its risk height correlation (p < 0.00001); Its representative example includes but not limited to: (a) contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQ ID NO:1,2,4,6,7,8,10,12,13,15,17,19,21,23,24,25 or 26, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:3,5,9,11,14,16,18,20 or 22; (c) contain coding and at least a portion of following sequence and have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:3,5,9,11,14,16,18,20 or 22, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In other exemplary; Said method comprises level or the functionally active that detects with at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or each expression product of 21 kind of HVI marker gene (hereinafter referred to as " the relevant HVI marker gene of secondary ") that have herpesvirus infection or associated conditions or its risk height correlation (p < 0.0001); Its representative example includes but not limited to: (a) contain and below arbitrary sequence have at least 50% (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQID NO:27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61 or 63, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:28,30,32,36,40,42,44,46,48,50,52,56,58,60,62 or 64; (c) contain coding and below at least a portion of arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:28,30,32,36,40,42,44,46,48,50,52,56,58,60,62 or 64, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In other exemplary; Said method comprises level or the functionally active that detects with at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or each expression product of 15 kind of HVI marker gene (hereinafter referred to as " three grades of relevant HVI marker gene ") that have herpesvirus infection or associated conditions or its risk medium relevant (p < 0.0003); Its representative example includes but not limited to: (a) contain and below arbitrary sequence have at least 50% (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQ ID NO:65,66,67,69,71,73,75,76,77,79,81,83,84,85 or 87, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:68,70,72,74,78,80,82,86 or 88; (c) contain coding and below at least a portion of arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:68,70,72,74,78,80,82,86 or 88, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In other exemplary; Said method comprises level or the functionally active that detects with each expression product of at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,16 or the 17 kind of HVI marker gene that have herpesvirus infection or associated conditions or its risk appropriate relevant (p < 0.06) (hereinafter referred to as " level Four be correlated with HVI marker gene "); Its representative example includes but not limited to: (a) contain and below arbitrary sequence have at least 50% (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQ ID NO:89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, or its complementary sequence; (b) contain the polynucleotide of nucleotide sequence that coding comprises the polypeptide of following arbitrary aminoacid sequence: SEQ ID NO:90,92,95,97,103,105,110,112 or 114; (c) contain coding and below at least a portion of arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of polypeptide of sequence similarity: SEQ ID NO:90,92,95,97,103,105,110,112 or 114, wherein said part contains 15 contiguous amino acid residues of this sequence at least; (d) containing at least can be under low preciseness, middle preciseness or high preciseness condition and the polynucleotide of the nucleotide sequence of (a) and (b), (c) said sequence or its complementary sequence hybridization.

In some embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least a kind of one-level of detection.In other embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 2 kinds of one-levels of detection.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of secondary expression product.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least 2 kinds of one-levels of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of secondary expression product.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least 2 kinds of relevant HVI marker gene of secondary expression products.

In some embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least a kind three grades relevant HVI marker gene expression products.In other embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least 2 kinds of one-levels of detection or the level or the functionally active of functionally active and at least a kind three grades relevant HVI marker gene expression products.In also having other embodiment, these methods comprise the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least 2 kinds three grades relevant HVI marker gene expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least 3 kinds three grades relevant HVI marker gene expression products.

In some embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of level Four expression product.In other embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least 2 kinds of one-levels of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of level Four expression product.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least 2 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least 3 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of one-level of detection or the level or the functionally active of functionally active and at least 4 kinds of relevant HVI marker gene of level Four expression products.

In some embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least a kind of secondary of detection.In other embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 2 kinds of secondarys of detection.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least a kind three grades relevant HVI marker gene expression products.In other embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least 2 kinds of secondarys of detection or the level or the functionally active of functionally active and at least a kind three grades relevant HVI marker gene expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 2 kinds three grades relevant HVI marker gene expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 3 kinds three grades relevant HVI marker gene expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 4 kinds three grades relevant HVI marker gene expression products.

In some embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of level Four expression product.In other embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least 2 kinds of secondarys of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of level Four expression product.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 2 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 3 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 4 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 5 kinds of relevant HVI marker gene of level Four expression products.

In some embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least a kind of secondary of detection.In other embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 2 kinds of secondarys of detection.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of Pyatyi expression product.In other embodiments, said method comprises the level of the relevant HVI marker gene expression product of at least 2 kinds of secondarys of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of Pyatyi expression product.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 2 kinds of relevant HVI marker gene of Pyatyi expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 3 kinds of relevant HVI marker gene of Pyatyi expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 4 kinds of relevant HVI marker gene of Pyatyi expression products.In also having other embodiment, said method comprises the level of the relevant HVI marker gene expression product of at least a kind of secondary of detection or the level or the functionally active of functionally active and at least 5 kinds of relevant HVI marker gene of Pyatyi expression products.

In some embodiments, said method comprises the level or the functionally active of at least a kind three grades relevant HVI marker gene expression products of detection.In other embodiments, said method comprises the level or the functionally active of at least 2 kinds three grades relevant HVI marker gene expression products of detection.In also having other embodiment, said method comprises the level of at least a kind three grades relevant HVI marker gene expression products of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of level Four expression product.In other embodiments, said method comprises the level of at least 2 kinds three grades relevant HVI marker gene expression products of detection or the level or the functionally active of functionally active and at least a kind of relevant HVI marker gene of level Four expression product.In also having other embodiment, said method comprises the level of at least a kind three grades relevant HVI marker gene expression products of detection or the level or the functionally active of functionally active and at least 2 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of at least a kind three grades relevant HVI marker gene expression products of detection or the level or the functionally active of functionally active and at least 3 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of at least a kind three grades relevant HVI marker gene expression products of detection or the level or the functionally active of functionally active and at least 4 kinds of relevant HVI marker gene of level Four expression products.In also having other embodiment, said method comprises the level of at least a kind three grades relevant HVI marker gene expression products of detection or the level or the functionally active of functionally active and at least 5 kinds of relevant HVI marker gene of level Four expression products.

In some embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least a kind of level Four of detection.In other embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 2 kinds of level Four of detection.In other embodiments, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 3 kinds of level Four of detection.In also having other embodiment, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 3 kinds of level Four of detection.In also having other embodiment, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 4 kinds of level Four of detection.In also having other embodiment, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 5 kinds of level Four of detection.In also having other embodiment, said method comprises the level or the functionally active of the relevant HVI marker gene expression product of at least 6 kinds of level Four of detection.

Biological sample preferably includes and should contain leukocytic blood, particularly peripheral blood.Said expression product should be selected from RNA molecule or polypeptide.In some embodiments, said expression product is identical with corresponding expression product.In other embodiments, the said expression product variant (for example, allele variant) that is corresponding expression product.

In certain embodiments; Said expression product or corresponding expression product be target RNA (for example; MRNA) or the DNA of this target RNA copy; Its horizontal available energy copies at least a nucleic probe of hybridizing with this target RNA or this DNA and detects under low preciseness condition, this nucleic probe contains 15 contiguous nucleotides of certain HVI mark polynucleotide at least.In these embodiments, this target RNA or its DNA are copied level or the abundance stdn by the DNA copy of the reference RNA that exists in the same sample or this reference RNA of detected level or abundance.Nucleic probe should be fixed on solid or the semi-solid upholder.In the exemplary of this type, this nucleic probe forms the part of nucleic probe space array.In some embodiments, detect the level that copies the bonded nucleic probe with this target RNA or this DNA through hybridization (for example adopting nucleic acid array).In other embodiments, detect the level that copies the bonded nucleic probe with this target RNA or this DNA through nucleic acid amplification (for example, adopting polymerase chain reaction (PCR)).In also having other embodiment, detect the level that copies the bonded nucleic probe with this target RNA or this DNA through the nucleicacidase protection test.

In other embodiments, said expression product or corresponding expression product are the target polypeptide, and its horizontal available energy and this target polypeptide the interactional at least a antigen binding molecules of immunity take place detect.In these embodiments, with the detected level of this target polypeptide or abundance by the reference polypeptide level standardization that exists in the same sample.Antigen binding molecules should be fixed on solid or the semi-solid upholder.In the exemplary of this type, this antigen binding molecules forms the part of antigen binding molecules space array.In some embodiments, with the level of immunoassay (for example utilizing ELISA) detection with this target polypeptide bonded antigen binding molecules.

In also having other embodiment, said expression product or corresponding expression product are the target polypeptide, and at least a substrate that its horizontal available energy and the reaction of this target polypeptide form reaction product detects.In these embodiments, with of the functionally active stdn of the detected functionally active of this target polypeptide by the reference polypeptide that exists in the same sample.

In some embodiments, available suitably comprise at least one with base station (base station) mutually a kind of system in the TS (end station) that joins of coupling carry out this diagnostic method.This base station should (a) be accepted data through network of communication from the TS; Wherein said data represented parameter value is detected or level of standardization or functionally active corresponding at least a expression product in the biological sample; (b) with said data with represent the detected or standardization of at least a corresponding expression product in the reference sample or the tentation data of functionally active to make comparisons; Thereby measure with the reference sample in the flat or functionally active of corresponding expression product water compare any difference of the level of this expression product or functionally active in this biological sample.The base station preferably can also diagnose whether have herpesvirus infection associated conditions or its degree.In these embodiments, the base station also can will diagnose indication to be passed to the TS through network of communication.

On the other hand; Broadly described method was monitored, is treated and control and has the animal that possibility causes the patient's condition of herpesvirus infection more than the present invention had considered to adopt, and the exemplary of the said patient's condition comprises: immunosuppression, new birth, stress or high-intensity training scheme.In these embodiments, use diagnostic method of the present invention often can effectively monitor the early stage generation or the associated conditions of herpesvirus infection, thus can the intervention of early treatment property and this illness of treatment.

On the other hand, the present invention provides treatment, prevention or suppresses herpesvirus infection in the object or the method for associated conditions.These methods generally comprise the unconventionality expression of at least a HVI marker gene in the detected object, and the medicine that gives this object significant quantity is with treatment or relief of symptoms, perhaps reverse or suppress this object generation herpesvirus infection or associated conditions.The representative example of this treatment or medicine includes but not limited to: the palliative supportive treatment of microbiotic, steroid and anti-inflammatory drug, intravenously (injection) liquid, vasoactive (medicine), damage or damage organ (for example; The oxygen supply of respiratory distress, the hypovolemia transfusion) and monitor vital organ closely.

On the other hand; The present invention provides this paper to be called the isolating polynucleotide of " HVI mark polynucleotide "; Said polynucleotide are selected from: (a) contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polynucleotide of nucleotide sequence of sequence homogeny: SEQ ID NO:1,12,23,24,25,26,33,34,37,38,65,66,75,76,83,84,93,98,99,100,101,106,107 or 108, or its complementary sequence; (b) contain the polynucleotide that comprise a following arbitrary sequence part: SEQ ID NO:1,12,23,24,25,26,33,34,37,38,65,66,75,76,83,84,93,98,99,100,101,106,107 or 108; Or its complementary sequence, wherein said part contains 15 contiguous nucleotides of this sequence or complementary sequence at least; (c) at least can be under low preciseness, middle preciseness or high preciseness condition and (a) or (b) polynucleotide of said sequence or its complementary sequence hybridization; (d) contain the polynucleotide of the part of following arbitrary sequence: SEQ ID NO:1,12,23,24,25,26,33,34,37,38,65,66,75,76,83,84,93,98,99,100,101,106,107 or 108; Or its complementary sequence, wherein said part contains 15 contiguous nucleotides of this sequence or complementary sequence and at least can be under low preciseness, middle preciseness or high preciseness condition and (a) and (b) or (c) said sequence or its complementary sequence hybridization at least.

Also having on the other hand, the present invention provide contain operability be connected in regulatory element more than the nucleic acid construct thing of broadly described polynucleotide, this construction can be operated in host cell.In certain embodiments, said construction is a carrier format, particularly expression vector.

Also having on the other hand, the present invention provides the isolating host cell that contains above broadly described nucleic acid construct thing or carrier.In some preferred embodiment, said host cell is selected from bacterial cell, yeast cell and insect cell.

Also having on the other hand, the present invention be provided for detecting whether exist in the nucleic acid more than the probe of broadly described polynucleotide.These probes generally comprise at least can under the low preciseness condition with more than the nucleotide sequence of broadly described multi-nucleotide hybrid.In some embodiments; Said probe is basically by constituting corresponding to nucleotide sequence at least a portion of arbitrary aminoacid sequence below the coding or with its complementary nucleotide sequence: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, wherein said part is to 15 Nucleotide of the youthful and the elderly.In other embodiments; Said probe contains the nucleotide sequence that can under low preciseness condition, hybridize with nucleotide sequence at least a portion of arbitrary aminoacid sequence below the coding at least: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, wherein said part is to 15 Nucleotide of the youthful and the elderly.In also having other embodiment; Said probe contains the nucleotide sequence that can under low preciseness condition, hybridize with at least a portion of following sequence at least: SEQ ID NO:1,2,4,6,7,8,10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, wherein said part is to 15 Nucleotide of the youthful and the elderly.The representative probe that is used to detect HVI mark polynucleotide of the present invention is shown in SEQID NO:145-2150 (seeing table 2).

In related fields, the present invention provide fixed above that at least a more than the solid or the semi-solid upholder of broadly described nucleic probe.In some embodiments, this solid or semi-solid upholder comprise fixing nucleic probe space array on it.

In others; The present invention provides this paper to be called the isolated polypeptide of " HVI labeling polypeptide "; Said polypeptide generally is selected from: (i) contain and more than the expression of polypeptides product of broadly described HVI marker gene have 50% at least (and at least 51%-at least 99% and between all integer per-cents) polypeptide of aminoacid sequence of sequence similarity; For example, particularly contain and below arbitrary sequence have 50% at least (and at least 51%-at least 99% and between all integer per-cents) the HVI marker gene of nucleotide sequence of sequence homogeny: SEQ ID NO:1,2,4,6,7,8,10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113; (ii) as (i) part of said polypeptide, wherein this part contains 5 contiguous amino acid residues of this polypeptide at least; (iii) contain and (i) at least 15 contiguous amino acid residues of said polypeptide have at least 30% similarity (and at least 31%-at least 99% and between all integer per-cents) the polypeptide of aminoacid sequence; (iv) contain can with (i), (ii) or (iii) said sequence the polypeptide that immune interactional aminoacid sequence takes place immune interactional antigen binding molecules takes place.

A further aspect of the present invention provides and can immune interactional antigen binding molecules take place with above broadly described HVI labeling polypeptide.

In related fields, the present invention provides solid or the semi-solid upholder of having fixed at least a above-mentioned antigen binding molecules above that.In some embodiments, this solid or semi-solid upholder comprise fixing antigen binding molecules space array on it.

The present invention provides broadly described HVI mark polynucleotide more than one or more in addition on the other hand, broadly described probe, broadly described HVI labeling polypeptide or the application of broadly described antigen binding molecules in the test kit that preparation diagnosis object herpesvirus infection associated conditions exists more than one or more more than one or more more than one or more.

Each side of the present invention relates to above broadly described diagnostic method, broadly described HVI mark polynucleotide more than one or more, broadly described probe more than one or more, more than one or more broadly described HVI labeling polypeptide or more than one or more broadly described antigen binding molecules be used for diagnosis and suffer from the herpesvirus infection associated conditions.Animal (vertebrates), Mammals, non-human mammal for example relate to the horse and the pet (for example, dog and cat) that bear a heavy burden or move (for example sports).

Each side of the present invention relates to animal (vertebrates), Mammals, non-human mammal, for example relates to the horse and the pet (for example, dog and cat) that bear a heavy burden or move (for example sports).

The accompanying drawing summary

Fig. 1 is the receptor operating curve (Receiver Operator Curve) of group 1 virus inoculation animal, has compared the genetic expression with other fate in the 2nd, 4 and 6 day.Utilize this check sensitivity and the specificity of genetic expression mark (signature) outstanding, TG-AUC surpasses 0.9.

Fig. 2 is when utilizing the selection gene of clinical infection animal, the diagram of receptor operating curve.Utilize the check sensitivity and the specificity of genetic expression mark outstanding.

Fig. 3 utilizes when regarding the selection gene of the animal with reactivity virus infection as the diagram of receptor operating curve.Utilize this check sensitivity and the specificity of genetic expression mark outstanding.

Fig. 4 is when utilizing all genes of clinical infection animal, the diagram of receptor operating curve.Utilize this check sensitivity and the specificity of allelic expression good.

Fig. 5 utilizes when regarding the selection gene with challenge virus infected animals as the diagram of receptor operating curve.Utilize this check sensitivity and the specificity of allelic expression good.

Fig. 6 shows that EHV-1 organizes the synoptic diagram of gene expression index (Log volume unit), serum EHVAb level (450nm absorbancy), date, fate (D=days) and the clinical sign of 1 young coltfoal.The children coltfoal is in inoculation on March 18th, 2003 (virus).The variation of gene expression index is corresponding to the clinical sign that exists, before 10-14 days of anti--EHV-1 antibody generation that this variation occurs in specific serum.

Fig. 7 is that the main ingredient of group 1 young coltfoal is analyzed synoptic diagram.These components are according to the fate mapping of inoculation after (virus).

Detailed Description Of The Invention

1. definition

Only if definition is arranged in addition, the same meaning of all technology used herein and scientific terminology and one skilled in the art's common sense of the present invention.Though the present invention is implemented or checked to any method similar or of equal value capable of using and described herein and material, preferred method as herein described and material.For the object of the invention has defined following term.

Article used herein " one " and " one " refer to that one or more (being at least one) this article is at phraseological object.For example, " element " refers to an element or a plurality of element.

This paper is used to describe the term " unconventionality expression " that the HVI marker gene is expressed; Refer to and take from health objects or do not receive certain HVI marker gene in the cell of herpesvirus infection object or the expression level of its variant; And/or with take from health objects or (for example do not have in tissue sample or the body fluid of object of simplexvirus certain HVI marker gene product; Transcript or polypeptide) higher or lower level compare this HVI marker gene over-expresses or express not enough.Specifically; If compare with the expression level of taking from health objects or not having certain HVI marker gene in cell, tissue or the humoral sample of the object of simplexvirus; This HVI marker gene is high at least by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; Or even high at least by about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%; Perhaps low at least about 10%, 20%, 30%40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%; Or even low at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, this HVI marker gene unconventionality expression then.

This paper is used to describe the term " unconventionality expression " that HVI mark polynucleotide are expressed; Refer to and take from health objects or (for example do not have the herpesvirus infection relative disease; The expression level of certain HVI mark polynucleotide or its variant in the cell of object no reactivity herpesvirus infection); And/or take from health objects or (for example do not have in tissue sample or the body fluid of object of herpesvirus infection disease certain HVI mark polynucleotide products; Transcript or polypeptide) higher or lower level compare this HVI mark polynucleotide over-expresses or express not enough.Specifically; If with take from health objects or do not have the expression level of certain HVI marker gene in cell, tissue or the humoral sample of the object of simplexvirus disease; And/or compare with the expression level of taking from health objects or not having certain HVI mark polynucleotide in tissue sample or the body fluid of object of simplexvirus disease; The expression level of these HVI mark polynucleotide is high at least by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; Or even high at least by about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%; Perhaps hang down about 10%, 20%, 30%40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99% at least; Or even low at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, this HVI mark polynucleotide unconventionality expression then.Abnormal gene expression in the immune system cell of the present invention, particularly circulating leukocyte can be derived from following two consecutive steps: (1) notes abnormalities, and the gene of expressing is diagnosed, prognosis is assessed with illness; (2) gene of clinical affirmation unconventionality expression.

(for example often organizing the cell or tissue sample according to following steps; Immune cell; Such as but not limited to white corpuscle) in receive those genes of remarkable rise or downward modulation (p < 0.06) and confirm the genetic expression that notes abnormalities: (a) keep the constant gene of constant and come stdn according to expression under normal and disease state; (b) adopt and to prevent that false-positive statistical method (for example, Holm and FDR judge) from explaining multivariate data, for example microarray data institute inherent false positive discovery.The technician of gene expression data analysis field know admissible data normalization other form and can material change's character of the present invention (for example MAS5, stable multicore sheet equalization (Robust multi chip averaging), multicore sheet equalization (GC Robust multi chip averaging) or Li Wong algorithm that GC is stable).With regard to diagnosis, the cell or tissue sample derives from one group usually and represents the true negative cell or tissue sample of illness interested and the true positives cell or tissue sample that a group is represented this illness.All other parameters or variable need suitably utilize identical animal usually and induce this animal that illness interested takes place and control in these groups, and for example age, geographical position, sex, motion health change with other normal biology.The technician in experimental design field knows and can adopt other method to control other parameter and variable and can material alterations character of the present invention.This method includes but not limited to: the co-variation amount (covariates) that randomization, blocking-up (blocking) and utilization are analyzed.With regard to prognosis, the cell or tissue sample derives from one group represent true positives cell or tissue sample that the true negative cell or tissue sample of illness interested and (behind the certain hour) subsequently represent this illness same group usually.All other parameters or variable need utilize identical animal usually in these groups; Induce those animals that illness interested takes place and control with changing the sample of taking from same animal in time, for example age, geographical position, sex, motion health change with other normal biology.With regard to assessment, the cell or tissue sample derive from usually one group of an end of the detected clinical parameter collection of illustrative plates relevant with representing illness interested with represent along the array that can detect clinical parameter collection of illustrative plates each point.Similarly, all other parameters or variable need suitably utilize identical animal usually and induce this animal that illness interested takes place and control in these groups, and for example age, geographical position, sex, motion health change with other normal biology.

In finding inventory according to the constant gene stdn in the cell or tissue after; Those genes that proof significantly raises or reduces; And analyze that interested situation is to make the gene of unconventionality expression those genes in correct diagnosis of at least 75% time or assessment illness in the clinical cell or tissue sample used in discover method, thus confirm clinical really with abnormal gene expression.Receptor operating curve (ROC) generally is a useful testing tool of carrying out this diagnosis.The technician of gene expression data analysis field knows that available other standardized method (for example MAS5, multicore sheet equalization or LiWong algorithm that stable multicore sheet equalization, GC is stable) replaces constant gene standardized method and can material alterations character of the present invention.In addition, the technician of gene expression data analysis field knows that can adopt many methods to measure those genes receives " significantly raising or downward modulation ".

" pact " refers to compare with amount, level, numerical value, numeral, frequency, per-cent, dimension, size, consumption, weight or the length of reference article, and 30,25,20,25,10,9,8,7,6,5,4,3,2 or 1% different amount, level, numerical value, numeral, frequency, per-cent, dimension, size, consumption, weight or length variations arranged at most.

Term " reactivity infection " uses with wide significance at this paper, comprise virus to host's intrusion, live away from home and/or breed, usually clinically maybe be obviously or maybe unconspicuous pathology symptom with one or more.Reactivity infects and comprises part, subclinical or temporary infection.Local infection can exist lastingly and expand to acute, subacute or chronic clinical infection or morbid state through propagation.When virus got into lymph or vascular system, local infection also can become general." reactivity infection " is often referred to host's immunity system by infectant activated Infection Status.

The amplified production of the target sequence that term " amplicon " refers to increase and/or the target sequence of amplification.In some other embodiment, " amplicon " can comprise probe or the primer sequence that is used to increase.

" antigen binding molecules " refers to target antigen is had the molecule of binding affinity.Will be appreciated that this term can extend to Tegeline, immunoglobulin fragment and NIg deutero-protein framework that demonstration has antigen-binding activity.

When term used herein " specificity combination ", " specific immunity interaction " etc. referred to certain antigen binding molecules, this term referred to can confirm in the heterogeneous population of protein and other biological products, to have this antigenic association reaction.Therefore, under specified immunoassay condition, said antigen binding molecules can combine with its specific antigen and not combine with other protein or the antigen that exists in obvious amount and the sample.Under this condition, combine and to select that this specific antigen is had specific antigen binding molecules by needs with certain antigenic specificity.For example, can prepare the antigen binding molecules to selected proteantigen, this molecular energy with this antigen but not in the sample other proteantigen combine.Can adopt various forms of immunoassay select can with the interactional antigen binding molecules of certain specified protein generation specific immunity.For example, conventional adopt solid phase ELISA immunoassay select can with the interactional monoclonal antibody of certain protein generation specific immunity.The immunoassay mode and the condition that can be used for measuring specific immune response property can be referring to Harlow and Lane, (1988), " antibody, laboratory manual " (Antibodies, A Laboratory Manual), cold spring port publication, New York.

" BA part " referred to keep the total length parental generation peptide of parental generation molecular activity or the part of polypeptide.Term used herein " BA part " comprises deletion mutant with parental generation molecular activity and the peptide that for example has at least about 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,40,50,60,70,80,90,100,120,150,300,400,500,600,700,800,900,1000 continuous amino acid residues.The recombinant nucleic acid technology of available standards obtains or adopts conventional liquid phase or solid phase synthesis technique to synthesize this type of part.For example; Can be with reference to publishing like Blackwell Scientific Publications, Atherton and Shephard institute are famous in the publication of " synthetic vaccine " (Synthetic Vaccines) by name that Nicholson compiled synthesizes or solid phase synthesis for the liquid phase described in " peptide synthesizes " (Peptide Synthesis) the 9th chapter.Perhaps, available proteolytic enzyme, for example endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease digestion peptide of the present invention or polypeptide prepare this type peptide.Can be through for example high performance liquid chromatography (HPLC) fragment that technological purifying digested.Also can adopt the recombinant nucleic acid technology to prepare this protein.

That term used herein " biological sample " refers to extract from animal, be untreated, handle, dilution or spissated sample.Biological sample can comprise biological fluid, for example whole blood, serum, blood plasma, saliva, urine, sweat, ascites, peritoneal fluid, synovial membrane liquid, amniotic fluid, cerebrospinal fluid, biopsy (sample) etc.In certain embodiments, biological sample is blood, particularly peripheral blood.

Should understand term used herein " cis acting sequence ", " cis-acting elements ", " cis regulatory region " or " regulatory region " or similar term and refer to when placing certain appropriate location that can express genetic sequence, can regulate, part is regulated any nucleotide sequence that this genetic sequence is expressed at least.Those skilled in the art will know that the cis regulatory region can transcribe or translate activation, silence, enhancing, inhibition on the level of back or change expression level and/or the cell type specificity and/or the development-specific of gene order.In certain embodiments of the invention, cis acting sequence is to strengthen or to stimulate certain can express the activation sequence that genetic sequence is expressed.

Only if needs are arranged in the literary composition in addition, the word in this specification sheets " comprises " and " containing " is interpreted as referring to comprise certain said step or element or one group of step or element, but does not get rid of any other step or element or one group of step or element.

" correspondence " or " corresponding to " refer to following polynucleotide: (a) all or part of of its nucleotide sequence that contains and certain reference polynucleotide sequence is substantially the same or complementary, or (b) its amino acid sequence coded is identical with certain peptide or proteinic aminoacid sequence.The scope of this phrase also comprises aminoacid sequence peptide or the polypeptide substantially the same with reference peptide or proteinic aminoacid sequence that contains.

In this paper treatment or prevent in the chapters and sections of certain disease; " significant quantity " refers to need the individuality of this treatment or prevention, this consumption can prevent effectively that the symptom of this illness from this symptom and/or the existing symptom of treatment taking place, suppressing with the part of single dose or successive doses the activity of this consumption (compound).Significant quantity according to wait to treat individual health with physical appearance, the assessment of formulation, medical condition of waiting to treat taxonomy group, the compsn of individuality is with other correlative factor and different.Hope that this consumption can confirm to have relative broad range with routine test.

Term " expression " or " genetic expression " refer to produce RNA information or RNA information are translated as protein or polypeptide.Adopting methods described herein to detect all types of genetic expressions is parts of the present invention.

" expression vector " refers to instruct the contained polynucleotide of this carrier to transcribe and suitably synthesizes the coded peptide of these polynucleotide or any autonomous genetic elements of polypeptide.Those skilled in the art will know that this expression vector.

Genomic any with all discontinuous coding regions of term used herein " gene " phalangeal cell and relevant non-coding and regulatory region thereof.5 ' and 3 ' the non-coding nucleotide sequence of regulating that adjoins is expressed in gene also refer to encode ORF, intron, the participation of concrete polypeptide.In this, gene also can contain and the natural relevant adjustment signal of certain given gene, for example promotor, enhanser, termination and/or polyadenylation signal, or allos adjustment signal.Dna sequence dna can be cDNA or genomic dna or its fragment.Can gene be introduced that to be maintained at karyomit(e) in the suitable carriers outer or be integrated into the host.

" high-density polynucleotide array " etc. refers to every cm ²At least those arrays that contain 400 different elements (feature).

Phrase " height distinctive hybridization conditions " refers to determine the hybridization conditions of a base mispairing.

This paper produces hybrid dna-DNA or hybrid dna-RNA with the pairing of " hybridization " expression complementary nucleotide sequence.Complementary base sequence is through basepairing rule those sequences that are mutually related.In DNA, A and T pairing, C and G pairing.In RNA, U and A pairing, C and G pairing.In this, term used herein " coupling " and " mispairing " refer to the hybridization possibility of pairing Nucleotide in the complementary nucleic acid chain.The Nucleotide of coupling can effectively be hybridized for example above-mentioned typical A-T and C-G base pairing.Mispairing is other combination that can not effectively hybridize Nucleotide.

Phrase " specific hybrid " etc. refers to that under the preciseness condition when certain specific nucleotide sequence was present in complicated DNA or the RNA mixture (for example, total cell), certain molecule only combined, forms duplex or hybridization with this sequence.

This paper mentions that " immunity interacts " comprises any interaction, reaction or other combining form of (particularly when one of said molecule is or simulates the immunity system component) between the molecule.

" immunologic function " or " immunoreactivity " refers to detect immunity system to external antigenic response capacity through standard test well known in the art.

The said material of " isolating " expression in fact or be substantially free of its under native state normally with component.For example, " isolating polynucleotide " used herein have referred to remove the polynucleotide of its side joint sequence under the native state, have for example removed the dna fragmentation that normally adjoins sequence with it.Perhaps, " isolating peptide " used herein or " isolated polypeptide " etc. refer to external with peptide or peptide molecule and its n cell environment and with it other cellular component of bonded be separated and/or purifying.Isolating polynucleotide, peptide or polypeptide can refer to that (being not limited to) is through separating and the native sequences of purifying or the sequence that produces through reorganization or compound method.

" marker gene " thus expression can be given the cell of expressing this marker gene that unique phenotype makes this transformant and other gene of cell phase region that does not contain this mark.But selectable marker gene is given and is equaled the characteristic ability according to the resistance of selectivity factor (for example, the treatment process of weedicide, microbiotic, radiation, heat or other destruction no transformed cells) is selected.The proterties that screenable marker gene (or reporter gene) is given can promptly be identified (for example, non-existent beta-Glucuronidase, luciferase or other enzymic activity in the non-transformed cell) through observing or check through " screening ".

" natural generation " used herein nucleic acid molecule refers to have the RNA or the dna molecular of the nucleotide sequence of natural generation.For example, the naturally occurring nucleic acid molecule protein of natural generation of encoding.

" available from " represent certain sample, for example cell extract or nucleic acid or polypeptide extract be separate from or derived from particular source.For example, can be directly from the biological fluid of object or tissue, separate and obtain extract.

Term used herein " oligonucleotide " refers to through phosphodiester bond continuous a plurality of nucleotide residues (deoxyribonucleotide or ribonucleotide; Or its relevant structural variant or synthetic analogues, comprise and contain the Nucleotide of modifying or replacing glycosyl group etc.) polymkeric substance (or its relevant structural variant or synthetic congener) that constitutes.Therefore; Although nucleotide residue and the connecting key between residue that term " oligonucleotide " is often referred to wherein are naturally occurring nucleotide polymers; But the scope that should know this term also comprises various congeners, includes but not limited to: PNAG3 (PNA), thiophosphatephosphorothioate, phosphorodithioate, seleno SULPHOSUCCINIC ACID ESTER (phosphoroselenoate), two seleno SULPHOSUCCINIC ACID ESTERs (phosphorodiselenoate), phosphoroanilothioate, phosphoraniladate, phosphoramidite, methyl-phosphonate, 2-O-methylribose nucleic acid etc.The definite size of this molecule is looked concrete application and difference.Oligonucleotide is the inferior groups of the polynucleotide of 200 of length or following base.Preferably long 10-60 the base of oligonucleotide, most preferably long 12,13,14,15,16,17,18,19 or 20-40 base.Though oligonucleotide can be double-stranded, for example is used to make up variant nucleic acid sequences, oligonucleotide is strand normally, for example probe.Oligonucleotide of the present invention can be that justice or antisense oligonucleotide are arranged.

Term " oligonucleotide arrays " refers to contain the substrate of oligonucleotide probe, has deposited different known arrays in the known discontinuous position of this substrate surface.For example, this substrate can be like U.S. Patent number 5,424,186 described bidimensional substrate forms.This substrate can be used for oligonucleotide (matrix) array of synthetic two-dimensional space addressing.Perhaps, this substrate is characterised in that being rolled into three-dimensional tubular configuration through the thin slice with two-dimensional plane forms tube array.This substrate also can be taked the microballoon or the pearl form that link to each other with optical fiber surface, and for example Chee etc. is in that kind described in the WO00/39587.These oligonucleotide arrays have two kinds of different elements at least, and its density is every cm ²At least 400 elements.In certain embodiments, the density of these arrays can be every cm ²About 500, at least one thousand, at least one ten thousand, at least ten ten thousand, at least one 1,000,000 or at least one ten million elements.For example, this substrate can be silicon or glass, has the thickness of slide glass or deckglass, perhaps can be made up of other synthetic polymer.When the method that on this substrate, makes an experiment comprises that light detects, the substrate of available printing opacity.This term also refers to the substrate of a probe array and a coupled formation wafer part.

Term used herein " operability connection " or " operability links to each other " expression place structure gene under the regulation and control of promotor, thus this gene transcription of may command and optional translation.When making up allogeneic promoter/structure gene combination; Usually preferably with genetic sequence or promotor be placed in be provided with the distance of genetic transcription initiation site is natural with it in this genetic sequence or promotor and its gene of regulating and control, the distance cardinal principle of the gene of promptly derive this genetic sequence or promotor is identical.The known in the art permission to this not loss of function apart from doing some changes.Similarly, the regulating and controlling sequence element with place preferred orientation this element capable of using of heterologous gene under its control in its natural setting, the location in its gene of promptly deriving is confirmed.

Term " pathogenic agent " this paper with its most widely implication use, but refer to the biology or the infectant of the diseases induced reaction of the living tissue cell of infection animal.

Term used herein " polynucleotide " or " nucleic acid " refer to mRNA, RNA, cRNA, cDNA or DNA.This term is often referred to the polymerized form of the Nucleotide of 10 bases of the youthful and the elderly, no matter is the modified forms of ribonucleotide or deoxyribonucleotide or these two types of Nucleotide.This term comprises strand or the double chain form of DNA.

Term " polynucleotide variant " and " variant " refer to show the polynucleotide that have the basic sequence homogeny with the reference polynucleotide sequence, or the polynucleotide that can under following preciseness condition, hybridize with reference sequence.These terms also comprise wherein has one or more Nucleotide to add or deletion, or with the substituted polynucleotide of different Nucleotide.In this, well knownly can make some change to said polynucleotide, comprise sudden change, interpolation, disappearance and replace, thereby make the polynucleotide of change still keep the biological function or the activity of these reference polynucleotide according to the reference polynucleotide.Term " polynucleotide variant " and " variant " also comprise the allele variant of natural generation.

" polypeptide ", " peptide " and " protein " refer to polymkeric substance and the variant and the synthetic congener of amino-acid residue in the interchangeable use of this paper.Therefore, these terms are applicable to that wherein one or more amino-acid residues are the aminoacid polymers of synthetic alpha-non-natural amino acid (for example, the chemical analog of corresponding natural amino acid) and the aminoacid polymers of natural generation.

Term " polypeptide variants " refer to through at least one amino-acid residue interpolation, disappearance or replacement and with other polypeptide of reference polypeptide phase region.In certain embodiments, one or more amino-acid residues of reference polypeptide can use different amino acid to replace.Be described below, more well known amino acid can be replaced with other amino acid with similar quality roughly and the activity (the conservative replacement) that can not change polypeptide.

" primer " expression can start primer extension product synthetic oligonucleotide in the presence of suitable polymerization agent when a chain with DNA matches.For making amplification efficiency maximum, the preferred strand of primer, but or can be double-stranded.Thereby primer answers sufficiently long can when polymerization agent exists, cause synthetic extension products.The length of primer depends on many factors, comprising: the temperature that use, is adopted, template reaction condition, other reagent and primer source.For example; The complicacy that depends on target sequence; 3 ' terminal comparable template sequence length weak point of primer can extend nucleic acid chains at least about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,50,75,100,150,200,300,400,500 to 1 base, though the extensible 3 ' end that exceeds template sequence of primer 5 ' tip length.In certain embodiments, primer can be big polynucleotide, and for example about 35 Nucleotide are to more than thousands of bases.Can select the primer with template sequence " basically complementary ", these primers of design can be with template hybridization and as the synthetic starting point." complementary basically " represented the complementary enough and target polynucleotide hybridization of this primer.Primer does not preferably contain mispairing with the template that design is hybridized with it, but this is optional.For example, can the incomplementarity nucleotide residue be connected 5 ' end of primer, and the rest part of this primer sequence and template are complementary.Perhaps, can be with inserting between the extension of incomplementarity nucleotide residue or incomplementarity nucleotide residue in the primer, prerequisite is this primer sequence and the required template of extension products that forms synthetic this primer with the complementary enough of the template sequence of its hybridization.

" probe " refer to can with particular sequence or subsequence or other part bonded molecule of another molecule.Except as otherwise noted, term " probe " is often referred to and can matches and another polynucleotide (often being called " target polynucleotide ") bonded polynucleotide probes through complementary base.The preciseness that depends on hybridization conditions, probe can combine to lack the complementary target polynucleotide of sufficient sequence with this probe.Probe is mark directly or indirectly, and its scope comprises primer.

Term used herein " recombination of polynucleotide " refers to make it become that occurring in nature is not normally found form and at the polynucleotide of external formation through operation nucleic acid.For example, recombination of polynucleotide can be taked the expression vector form.This expression vector generally comprises operability and is connected in transcribing of this nucleotide sequence and translates adjusting nucleic acid.

Recombinant technology is adopted in " recombinant polypeptide " expression, promptly expresses the polypeptide for preparing through recon or synthetic polynucleotide.

" regulatory element " or " adjusting sequence " is illustrated in and expresses the required nucleotide sequence of the continuous encoding sequence of operability in the concrete host cell (for example, DNA).For example, be applicable to that the adjusting sequence of prokaryotic cell prokaryocyte comprises promotor and optional cis acting sequence, like operon sequence and ribosome bind site.Be applicable to that eukaryotic regulating and controlling sequence comprises the leading and tailer sequence of promotor, polyadenylation signal, transcriptional enhancer, translational enhancer, regulating mRNA stability, and the targeting sequencing that makes interior compartment of the polynucleotide encoding product targeted cells of transcribing or target born of the same parents external environment.

Term used herein " sequence homogeny " refer to two sequences on comparison window nucleotide pair Nucleotide or amino acid to amino acid whose homogeny degree.Therefore; " sequence homogeny per-cent " can calculate through following steps: the sequences that on comparison window, compare two best comparisons; (for example measure nucleotide base identical in the two sequences; A, T, C, G, I) or identical amino-acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) position number produce the position number of coupling, with the matched position number (promptly divided by total positional number of comparison window; Window size), again the result multiply by 100 and obtain sequence homogeny per-cent.Be the object of the invention, should " sequence homogeny " be interpreted as with DNASIS computer program (2.5 editions of existing windows; Available from Hitachi Software engineering Co., Ltd., South San Francisco, California, the U.S.), adopt the standard of using in the appended RM of this software to give tacit consent to " match-percentage " that (parameter) calculated.

Like 3 definition of following table, " similarity " refers to identical or constitutes conservative substituted amino acid percentage ratio.Sequence comparison program capable of using, for example GAP (Deveraux etc., 1984, Nucleic Acids Research, 12,387-395) mensuration similarity.Length and sequence similarity described herein or basically different sequences can be in this way in arrangement, insert the room and come comparison, this room can be confirmed through the used comparison algorithm of for example GAP.

The term that is used to describe the serial correlation between two or above polynucleotide or the polypeptide comprises " reference sequence ", " comparison window ", " sequence homogeny ", " sequence homogeny per-cent " and " substantially the same ".At least 12 of the length of " reference sequence ", but more common 15-18, often at least 25 monomeric units comprise Nucleotide and amino-acid residue.Because two polynucleotide respectively contain similar between (1) two a polynucleotide sequence (promptly; Be the part of whole polynucleotide sequence); Article (2) two, a differentiated sequence between the polynucleotide sequence, then general through in " comparison window " thus go up that the sequence of these two polynucleotide is relatively identified and relatively regional area sequence similarity two (more than) carry out sequence between the polynucleotide and compare." comparison window " is to contain at least 6; Usually about 50-is about 100; Notional fragment of about 150 continuous positions of more common about 100-, wherein after the best comparison of certain sequence and reference sequence, this sequence is made comparisons with the reference sequence that contains the similar number continuous position.Be the best comparison of (carrying out) two sequences, compare that comparison window can comprise about 20% or following interpolation or disappearance (that is room) with reference sequence (do not comprise and add or disappearance).Be the comparison comparison window; Can pass through computingmachine execution algorithm (GAP, BESTFIT, FASTA and TFASTA, Wisconsin Genetics Software Package Release7.0, GeneticsComputer Group; 575Science Drive Madison; WI, the U.S.), or compare (promptly causing highest homology property per-cent on the comparison window) through the best that one of range estimation and selected the whole bag of tricks produce and come all sequences are carried out the best comparison.Also can be like Altschul etc., 1997, Nucl.Acids Res., 25:3389 is said, compares through BLAST family program.Visible Ausubel etc., the 15th chapter of " up-to-date molecular biology method " (Current Protocols in Molecular Biology), Unit the 19.3rd, John Wiley&Sons Inc, 1994-1998 are detailed in sequential analysis.

Refer to any object, particularly vertebrate subject of needs treatment or prevention at term " object ", " individuality " or " patient " of the interchangeable use of this paper, even more refer in particular to mammalian object.Suitable vertebrates includes but not limited in the scope of the invention: primate, birds, domestic animal are (for example; Sheep, cow, horse, donkey, pig), the laboratory inspection animal (for example; Rabbit, mouse, rat, cavy, hamster), companion animals (for example; Cat, dog) and the wildlife (for example, fox, deer, wild dog) that catches.Preferably to as if need the related indication animal of treatment or prophylaxis of herpes viral infections or its, particularly horse class animal.However, it should be understood that above term has not hinted that symptom exists.

The phrase of this paper " similar basically avidity " refer to target sequence under selected one group of preciseness condition with the complementary of them or complementary oligonucleotide probe hybridization intensity is similar and can detect basically.

Term used herein " template " refers to be used for producing and the nucleic acid that is somebody's turn to do " template " chain complementary nucleic acid chains.Template can be RNA and/or DNA, and its complementary strand also can be RNA and/or DNA.In certain embodiments, complementary strand can comprise all or part of complementary sequence of this " template ", and/or can comprise sudden change, thereby it is not the complete complementary strand of this " template ".In detection as herein described test and other test known in the art, not with the complete complementary chain of template strand also can with the template strand specific hybrid, this complementary strand that can be used for detecting test is a part of the present invention.

Term " conversion " expression changes certain organism through introducing external source or endogenous nucleic acid, for example the genotype of bacterium, yeast, Mammals, birds, Reptilia, fish or plant.

Term " treatment " expression therapeutic and prophylactic treatment.

The polynucleotide molecule of certain polynucleotide can inserted or be cloned into to " carrier " expression therein, and suitable carriers can be derived from, the dna molecular of plasmid, bacteriophage, yeast, virus, Mammals, birds, Reptilia or fish for example.Carrier preferably contains the restriction site of one or more uniquenesses and can comprise self-replicating in target cell or tissue or its daughter cell or the tissue at the host cell of confirming, thereby or can integrate reproducible sequence of cloning with the host cell gene group of confirming.Therefore, carrier can be the carrier of self-replicating, the carrier that promptly exists with the outer entity of karyomit(e), and it duplicates and is independent of THE REPLICATION OF CHROMOSOME, for example linear or closed loop plasmid, extra-chromosomal element, minichromosomes or artificial chromosome.Carrier can contain the element of guaranteeing self-replication.Perhaps, it can be integrated in the genome and duplicates with the karyomit(e) that is integrated into after carrier is introduced host cell.Carrier system can comprise a kind of carrier or plasmid, contains two or more carriers or the plasmid of the total DNA that introduces the host cell gene group together, or transposon.The selection carrier generally depends on this carrier and waits to introduce the consistency of the host cell of this carrier.Carrier also can comprise selective marker, for example can be used for selecting the antibiotics resistance gene of suitable transformant.The example of the known this resistant gene of those skilled in the art.

Term " wild-type " and " normally " interchangeable use refer to the characteristic phenotype of the species of most of natural generations, and its opposite example is a mutation type surface.

2. abbreviation

Abbreviation below using in the specification sheets:

Nt=Nucleotide

Many Nucleotide of nts=

Aa=amino acid

Kb=kilobase or kilobase are right

The kDa=kilodalton

D=day

H=hour

S=second

3. the mark of herpesvirus infection and application thereof

The present invention relates to herpesvirus infection, particularly early detection, diagnosis, monitoring or the prognosis of EHV infection or its associated conditions.The invention discloses the cell of the object that has infected simplexvirus; Hemocyte particularly; More specifically be alternative (surrogate) mark of herpesvirus infection in the PBC, the form of said mark is the RNA molecule of particular sequence or the polypeptide of these RNA developed by molecule.These marks are indexs of herpesvirus infection; With compare in normal subjects or the expression do not had in the herpesvirus infection object; Diagnosable this tested object generation herpesvirus infection, particularly reactivity herpesvirus infection when their differential expressions, or have risk.The prior art in field is compared therewith, thisly is marked with many advantages.Utilize in the preferred embodiment that peripheral blood analyzes at some, possibly before detecting serum antibody, promptly diagnose herpesvirus infection.

It seems that nucleotide sequence as herein described can be used for the various Application Areass of simplexvirus detection, diagnosis, prognosis and treatment.In this paper scope these examples of applications comprise utilize primer amplified HVI mark, through with oligonucleotide probe hybridization, isolating nucleic acid is mixed carrier, makes the expression of nucleic acid that mixes carrier for RNA and protein and make the immunology reagent colour development corresponding to the coded product of this mark detect the HVI mark.

The HVI mark of being identified and then can be used for designs specificity oligonucleotide probe and primer.This probe and primer can be can with any length of the marker gene sequence-specific identified hybridization; It is about 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,50,75,100,150,200,300,400,500 Nucleotide at least, and probe is the total length of the marker gene sequence that this paper identifies.Thereby also can comprising other sequence at its 5 ' and/or 3 ' end, probe can extend beyond the target sequence with its hybridization.

When the coupling nucleic acid amplification method, the transcript of these probes and primer ability real-time analysis biological sample (for example, peripheral blood sample) certification mark gene or detection or quantitatively determined marker gene.This method comprises any method or the technology that is used to duplicate or increase the copy number or the content of target nucleic acid or its complementary sequence known in the art or described herein.

The mark of being identified also can be used for identifying the full-length gene order with the isolation of genomic DNA library, comprises the regulatory element of genetic expression, is preferably the element in (but being not limited to) source, Malaysia.The cDNA sequence that this paper identifies can be used as hybridization probe and comes the screening genome dna library with routine techniques.In case identify portion gene group clone, can be through " karyomit(e) walking " (being also referred to as " overlapping hybridization "), employing, for example Chinault and Carbon (1979, Gene, 5:111-126) described method is separated full-length gene.Obtain portion gene group clone in case utilize the cDNA hybridization probe to separate; Being positioned near this portion gene group clone two non-replicating sections terminal or it can be used as hybridization probe and does further genomic library screening, the final whole gene order that obtains HVI mark interested of separating.Should know available total length as herein described or Partial cDNA Sequence or short EST (ETS), for example adopt Sambrook etc., (" molecular cloning. laboratory manual " (MOLECULAR CLONING.A LABORATORY MANUAL); (cold spring port press; 1989) and Ausubel etc., (" up-to-date molecular biology method " (CURRENTPROTOCOLS IN MOLECULAR BIOLOGY), John Wiley&Sons; Inc., 1994) described standard technique obtains full-length gene.In addition, can adopt disclosedly, for example above, utilize said sequence to identify and obtain full length cDNA sequence with separating with reference to the standard technique described in the teaching material.Can utilize these technical evaluation and isolating sequence, adopt detection method described herein to detect the HVI marker gene, these sequences are parts of the present invention.

Those of ordinary skills can select some sections to be used for various detections, diagnosis or method of prognosis as the present invention's part from the marker gene of being identified; Vector construct; Preparation antigen binding molecules, test kit and/or any embodiment described herein.The marker gene sequence that the present invention preferably uses is SEQID NO:1; 2; 4; 6; 7; 8; 10; 12; 13; 15; 17; 19; 21; 23; 24; 25; 26; 27; 29; 31; 33; 34; 35; 37; 38; 39; 41; 43; 45; 47; 49; 51; 53; 55; 57; 59; 61; 63; 65; 66; 67; 69; 71; 73; 75; 76; 77; 79; 81; 83; 84; 85; 87; 89; 91; 93; 94; 96; 98; 99; 100; 101; 102; 104; 106; 107; 108; 109; Sequence shown in 111 or 113.

3.1 nucleic acid molecule of the present invention

Of embodiment and table 1, this paper provides and has passed through GeneChip ^TM63 kinds of simplexvirus marks (that is 63 kinds of simplexvirus marker gene) that analysis identifies from the blood of normal horses and the horses with EHV clinical evidence.In these 63 kinds of marker gene, 44 kinds have the total length or the encoding sequence of total length basically, and all the other 21 kinds have partial sequence information in their 5 ' and 3 ' terminal one or both ends.The HVI marker gene of being identified comprises 21 kinds of former not horse genes of characterized.

According to the present invention, find that isolated nucleic acid sequences described herein itself can be used as hybridization probe or amplimer.These nucleic acid can be used for, and for example diagnostic is assessed biological sample or is used to clone full-length cDNA or its corresponding genomic clone.In certain embodiments, these probes and primer provide length to be enough to and to mention from the RNA of biological sample or the oligonucleotide of DNA sample specific hybrid.About usually 10-20 the Nucleotide of these sequences is long, but can be longer.Some embodiments needs long sequence, for example about 30,40,50,100,500 Nucleotide and even total length nearly.

The present invention has considered to have following SEQ ID NO:1; 2; 4; 6; 7; 8; 10; 12; 13; 15; 17; 19; 21; 23; 24; 25; 26; 27; 29; 31; 33; 34; 35; 37; 38; 39; 41; 43; 45; 47; 49; 51; 53; 55; 57; 59; 61; 63; 65; 66; 67; 69; 71; 73; 75; 76; 77; 79; 81; 83; 84; 85; 87; 89; 91; 93; 94; 96; 98; 99; 100; 101; 102; 104; 106; 107; 108; 109; About 10 of arbitrary sequence shown in 111 or 113; 15; 17; 20; 30; 40; 50; 60; The nucleic acid molecule that adjoins the extension of 75 or 100 or 500 Nucleotide.The present invention also considered and above-mentioned sequence complementary, can be under high preciseness condition bonded molecule with it.These probes can be used for various hybridization embodiments, for example Southern and Northern trace.In some instances, the present invention considers to use the probe that can not damage their efficient diagnosis herpesvirus infection abilities with multiple target sequence hybridization.In a word, it is considered herein that the expression that reagent that hybridization probe described herein both can be used as solution hybridization (in PCR) detects corresponding gene also can be used in the solid phase embodiment.

Various probes and primer have been designed around said nucleotide sequence.For example, in certain embodiments, the sequence that is used for designing probe and primer can comprise the repetition extension (poly-A tail) of the adenine nucleotide of RNA two ends that are connected in institute's identifying mark gene usually.In other embodiments, those of ordinary skills possibly think that some section more is applicable to said detection method, so can probe and primer be specifically designed as these or other section that does not comprise institute's identifying mark gene.In any situation, those of ordinary skill all can be selected primer or probe sequence for selected Application Areas.The exemplary probe sequence that is used to detect the HVI marker gene is seen table 2.

Primer can be two strands or single stranded form, though single stranded form is more desirable.Can probe design be need not to be used for amplification method for combining (although possibly cause) with target DNA or RNA.In certain embodiments, available radioactive substance ( ³²P, ¹⁴C, ³⁵S, ³H or other affinity tag), fluorophore (for example rhodamine, resorcinolphthalein) or chemiluminescent labels (for example, luciferase) these probes of mark or primer.

The present invention provides cDNA sequence and the EST or the Partial cDNA Sequence of the total length basically that can be used as the EHV mark.However, it should be understood that this paper content is not limited to these disclosed sequences, particularly to comprise at least can with the isolating nucleic acid of the variant of the nucleic acid that contains disclosed sequence or these nucleic acid hybridization.For example, the partial sequence of available nucleic acid is identified gene or its deutero-full-length gene group or the cDNA clone of structurally associated.The cDNA of the target sequence that can be used as above-mentioned probe known in the art and the preparation method of genomic library (referring to, Sambrook etc. for example, 1989, the same and Ausubel etc., 1994, the same).All this nucleic acid and specific nucleic acid molecule as herein described are referred to as " simplexvirus mark polynucleotide " or " HVI mark polynucleotide ".In addition, scope of the present invention comprises the expression product (that is, rna transcription thing and polypeptide) of the isolated or purified of HVI mark polynucleotide.

Therefore, the present invention includes nucleic acid isolating or purifying basically or protein compsn." isolating " or " purifying " nucleic acid molecule or protein, or its BA partly is not contain the natural together normal or interactional component with it found in the environment that exists of this nucleic acid molecule or protein basically or in itself.Therefore, it is substantially free of other cellular material or medium component when the polynucleotide of isolated or purified or polypeptide produce through recombinant technology, perhaps when they are substantially free of precursor or other chemical substance during with chemosynthesis." isolating " polynucleotide should not contain the sequence (that is, being positioned at 5 ' and 3 ' terminal sequence of these polynucleotide) of natural these polynucleotide of side joint in the genomic dna of the organism that produces these polynucleotide, particularly proteinic encoding sequence.For example, in various embodiments, isolating HVI mark polynucleotide can contain the nucleotide sequence of natural these polynucleotide of side joint in the genomic dna of cell of these polynucleotide of generation that are less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb.The polypeptide that is substantially free of cellular material comprises the protein articles that contains the contaminating protein matter that is less than about 30%, 20%, 10%, 5% (with dry weight basis).When protein of the present invention or its BA were partly used recombinant methods, substratum should contain precursor or the non-proteins of interest materialization material that is less than about 30%, 20%, 10% or 5% (with dry weight basis).

The present invention also comprises the total length of HVI mark polynucleotide or the each several part of full length nucleotide sequence basically, perhaps the DNA of their transcript or these transcripts copy.The each several part codified polypeptide portion of HVI labeled nucleotide sequence or keep the section of this natural polypeptides BA.Perhaps, the each several part that can be used as the HVI labeled nucleotide sequence of hybridization probe is not encoded usually and is kept the aminoacid sequence of this BA.Therefore; The each several part of HVI labeled nucleotide sequence contains about 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,40,50,60,80,90,100 Nucleotide of the nucleotide sequence of code book invention HVI labeling polypeptide at least, perhaps almost reaches its total length.

The part of the HVI labeled nucleotide sequence of the BA part of code book invention HVI labeling polypeptide at least about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,40,50,60,70,80,90,100,120,150,300,400,500,600,700,800,900 or 1000 in the codified total length HVI labeling polypeptide, or even at least about 2000,3000,4000 or 5000 continuous amino acid residues, perhaps almost up to its amino acid sum.The each several part that can be used as the HVI labeled nucleotide sequence of hybridization probe or PCR primer generally need not the to encode BA part of HVI labeling polypeptide.

Therefore, the BA part of a part of codified HVI labeling polypeptide of HVI labeled nucleotide sequence, perhaps it can be the fragment that in standard method known in the art, is used as hybridization probe or PCR primer.Can be through separating the part of a HVI labeled nucleotide sequence of the present invention, the part (activity) expressing the part (for example passing through in-vitro recombination expression) of its coded HVI labeling polypeptide and assess this coded HVI labeling polypeptide prepares the BA part of this HVI labeling polypeptide.Nucleic acid molecule as HVI labeled nucleotide sequence each several part contains about 15,16,17,18,19,20,25,30,50,75,100,150,200,250,300,350,400,450,500,550,600 or 650 Nucleotide in the total length HVI labeled nucleotide sequence at least, perhaps almost up to its Nucleotide sum.

The present invention has also considered the variant of HVI labeled nucleotide sequence.The nucleic acid variant can be natural generation, for example allele variant (same locus), homologue (different genes seat) and directly to homologue (different organism) or can be that non-natural produces.Can adopt the Protocols in Molecular Biology of knowing, polymerase chain reaction for example known in the art (PCR) and hybridization technique are identified the variant of natural generation, for example said these variants.Can pass through induced-mutation technique, comprise that the technology that is applicable to polynucleotide, cell or organism prepares the variant that non-natural produces.These variants can contain Nucleotide replacement, disappearance, inversion and insertion.Coding can morph with non-coding region.Variation both can cause conservative property also can cause non-conservation aminoacid replacement (comparing with coded product).With regard to nucleotide sequence, examples of conservative variations comprise because of the genetic codon degeneracy still can code book those (nucleic acid) sequences of aminoacid sequence of one of invention HVI labeling polypeptide.The variant nucleotide sequence also comprises synthetic generation but still the nucleotide sequence of ability code book invention HVI labeling polypeptide, those sequences that for example produce through site-directed mutagenesis.Generally; The variant and this specific nucleotide sequence that utilize default parameters to be measured to the present invention's specific nucleotide sequence through sequence alignment program as herein described have about 30%, 40%, 50%, 55%, 60%, 65%, 70% at least; Generally at least about 75%, 80%, 85%; More than preferred about 90%-95%, the sequence homogeny more than 98% more preferably from about.

HVI labeled nucleotide sequence of the present invention can be used for separating other organism, particularly corresponding sequence of the horse of other Mammals, especially other kind and allelic variant sequence.The hybridizing method of nucleotide sequence is not difficult in this area to obtain.Can separate the encoding sequence that obtains other organism according to it with the sequence homogeny of encoding sequence described herein through knowing technology.In these technology, can the known coded sequence of all or part be used as probe, with cloned genes group dna fragmentation or middle other HVI marker coding sequence selective cross that exists of cDNA segment group (that is, genome or cDNA library) of selected organism.Therefore, the present invention also consider under following preciseness condition can with the polynucleotide of said HVI marker gene nucleotide sequence or its complementary sequence hybridization.The condition of having described hybridization and washing " hybridized " in term used herein under low preciseness, medium preciseness, high preciseness or high preciseness condition.Carry out (1998, the same) such as the visible Ausubel of guide of hybridization, the 6.3.1-6.3.6 part.Described water-based and non-aqueous method in this reference, the two is all available.The low preciseness condition that this paper mentions comprises at least about 1%-at least about the methane amide of 15%v/v with at least about the salt of 1-at least about 2M, 42 ℃ of hybridization, and at least about the salt of 1-at least about 2M, and 42 ℃ of washings.Low preciseness condition also can comprise 1% bovine serum albumin (BSA), 1mM EDTA, 0.5M NaHPO ₄(pH7.2), 7%SDS, 65 ℃ of hybridization, with (i) with 2 * SSC, 0.1%SDS; Or (ii) use 0.5%BSA, 1mM EDTA, 40mM NaHPO ₄(pH7.2), 5%SDS, room temperature washing.An embodiment of low preciseness condition comprises about 45 ℃ of hybridization in 6 * sodium chloride/sodium citrate (SSC), then at least 50 ℃ (wash temperature of hanging down the preciseness condition can be increased to 55 ℃) with 0.2 * SSC, the 0.1%SDS washed twice.Medium preciseness condition comprises at least about 16-at least about the methane amide of 30%v/v with at least about the salt of 0.5-at least about 0.9M, 42 ℃ of hybridization, and at least about the salt of 0.1-at least about 0.2M, and 55 ℃ of washings.Medium preciseness condition also can comprise 1% bovine serum albumin (BSA), 1mM EDTA, 0.5M NaHPO ₄(pH7.2), 7%SDS, 65 ℃ of hybridization, with (i) with 2 * SSC, 0.1%SDS; Or (ii) use 0.5%BSA, 1mM EDTA, 40mM NaHPO ₄(pH7.2), 5%SDS, 60-65 ℃ of washing.An embodiment of medium preciseness condition comprises that about 45 ℃ hybridize in 6 * SSC, 60 ℃ with 0.2 * SSC then, more than the 0.1%SDS washing once.High preciseness condition comprises that at least about 31-at least about the methane amide of 50%v/v and the salt of the about 0.15M of about 0.01-, 42 ℃ of hybridization are with the salt of the about 0.02M of the about 0.01-of usefulness, 55 ℃ of washings.High preciseness condition also can comprise 1%BSA, 1mM EDTA, 0.5M NaHPO ₄(pH7.2), 7%SDS, 65 ℃ of hybridization, with (i) with 0.2 * SSC, 0.1%SDS; Or (ii) use 0.5%BSA, 1mM EDTA, 40mM NaHPO ₄(pH7.2), 1%SDS, temperature washing more than 65 ℃.An embodiment of high preciseness condition comprises that about 45 ℃ hybridize in 6 * SSC, 65 ℃ with 0.2 * SSC then, more than the 0.1%SDS washing once.

In certain embodiments, the HVI labeling polypeptide is by can be under high preciseness condition and the polynucleotide encoding of said nucleotide sequence hybridization.An embodiment of high preciseness condition comprises that 65 ℃ hybridize in 0.5M sodium phosphate, 7%SDS, 65 ℃ with 0.2 * SSC then, more than the 1%SDS washing once.

Well known other preciseness condition, the technician knows can control the specificity that various factors is optimized hybridization.The preciseness that can optimize final washing step is to guarantee highly hybridization.Specific example can be referring to Ausubel etc., and is the same, 2.10.1-2.10.16 page or leaf and Sambrook etc., (1989, the same), 1.101-1.104 part.

Though generally under about 42 ℃-68 ℃ temperature, carry out the preciseness washing, those skilled in the art will know that other temperature also is suitable for the preciseness condition.For forming DNA-DNA hybridization, maximum hybrid rate generally occurs in and compares T _mLow about 20 ℃-25 ℃ temperature.Well known T _mBe melting temperature(Tm), or two dissociated temperature of complementary polynucleotide sequence.Well known estimation T _mMethod (referring to Ausubel etc., the same, 2.10.8 page or leaf).Generally, the double-helical T of optimum matching of following formula prediction DNA capable of using _mApproximation:

T _m=81.5+16.6 (log10M)+0.41 (%G+C)-0.63 (% methane amide)-(600/ length)

Wherein: M is Na ⁺Concentration, preferred 0.01-0.4 mole; %G+C is the per-cent that guanosine and cytidine base sum account for base sum, its scope 30% and 75%G+C between; The % methane amide is the methane amide percentage of by volume; Length is the number of base pair in the dna double spiral.Mismatched bases is to the every rising 1% of number, the T of duplex DNA at random _mReduce about 1 ℃.High preciseness washing is usually at T _mCarry out in the time of-15 ℃, perhaps medium preciseness is at T _m-30 ℃ are carried out.

In an example of hybridizing method; The film (for example, nitrocellulose filter or nylon membrane) that will contain fixed dna is hybridized in containing the hybridization buffer of label probe (50% deionized formamide, 5 * SSC, 5 * Denhardt solution (0.1%ficoll, 0.1% Vinylpyrrolidone polymer and 0.1% bovine serum albumin), 0.1%SDS and 200mg/mL denatured salmon sperm dna) for 42 ℃ and is spent the night.Then (promptly with twice of medium this film of preciseness continuous washing; 45 ℃ with 2 * SSC, 0.1%SDS washing 15 minutes; 50 ℃ with 2 * SSC, 0.1%SDS washing 15 minutes then); Again with twice of higher preciseness continuous washing (that is, 55 ℃ with 0.2 * SSC, 0.1%SDS washing 12 minutes, then 65-68 ℃ with 0.2 * SSC, 0.1%SDS solution washing 12 minutes).

3.2 polypeptide of the present invention

The present invention has also considered the full-length polypeptide of HVI marker gene coding of the present invention and the BA part of those polypeptides, is referred to as " herpesvirus infection mark polynucleotide " or " HVI labeling polypeptide " at this paper.The BA of total length HVI labeling polypeptide partly comprise be about 6,8,10,12,14,16,18,20,25,30,40,50,60 amino-acid residues at least have an immunity active part that interacts.For example, the immunity interaction fragment that the present invention considered is to 6 of the youthful and the elderlys, preferred at least 8 amino-acid residues, thus they can cause immunoreation and produce and can immune interactional antigen binding molecules take place with HVI labeling polypeptide of the present invention in animal.The relevant HVI labeling polypeptide of structure and/or function in available this other Mammals of antigen binding molecules screening, particularly horse class Mammals.The each several part of total length HVI labeling polypeptide generally possibly participated in certain interaction, for example intramolecularly or molecular interaction.Molecular interaction can be specificity binding interactions or enzymatic interaction (for example said interaction can be an instantaneity, can form or interrupt certain covalent linkage).The BA of total length HVI labeling polypeptide partly comprises the aminoacid sequence that contains with certain (supposition) total length HVI labeling polypeptide; Aminoacid sequence shown in for example is enough similar or derived from the peptide of the aminoacid sequence of this aminoacid sequence: SEQ ID NO:3,5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, these sequences contain a kind of activity that the amino acid that is less than total length HVI labeling polypeptide also shows this polypeptide at least.BA part comprises usually and has total length HVI labeling polypeptide a kind of active structures territory or motif at least.The BA part of total length HVI labeling polypeptide can be long; For example 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,40,50,60,70,80,90,100,120,150,300,400,500,600,700,800,900 or 1000, perhaps even at least about the polypeptide of 2000,3000,4000 or 5000 above amino-acid residues.Said part should be to contain to be not less than its about 1%, 10%, 25%, 50% active " the BA part " of full-length polypeptide of deriving.

The present invention has also considered variant HVI labeling polypeptide." variant " polypeptide comprises in the following manner from natural protein deutero-protein: delete (being called brachymemma) or add one or more amino acid at the N-of this natural protein end and/or C-end; In the one or more site deletion of this natural protein or add one or more amino acid; Perhaps the one or more site at this natural protein replace one or more amino acid.Variant proteins of the present invention has BA, and promptly they still have the required BA of this natural protein.For example, genetic polymorphism or manual operation can form this variant.Utilize default parameters to be measured to the BA variant of natural HVI labelled protein of the present invention and the aminoacid sequence of this natural protein can have at least 40%, 50%, 60%, 70% through sequence alignment program as herein described; Generally at least 75%, 80%, 85%; More than preferred about 90%-95%, the sequence similarity more than 98% more preferably from about.The proteinic BA variant of the present invention and this protein generally can have nearly 1000,500,400,300,200,100,50 or 20 amino-acid residues, perhaps should lack to 1-15 amino-acid residue, few to 1-10 (for example 6-10), lack to 5, lack to 4,3,2 perhaps even 1 amino-acid residue difference.

HVI labeling polypeptide of the present invention be can change in every way, aminoacid replacement, disappearance, brachymemma and insertion comprised.This area generally knows that this working method.The aminoacid sequence variant that for example, can prepare the HVI labelled protein through dna mutation.The method that well known mutagenesis and nucleotide sequence change.Referring to, for example Kunkel (1985, Proc.Natl.Acad.Sci.USA, 82:488-492), Kunkel etc., (1987, MethodsinEnzymol., 154:367-382), U.S. Patent number 4,873,192; Watson, J.D. etc., the reference that quote (" molecular biology of gene " (Molecular Biology of the Gene), the 4th edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and they.The guide that suitably carries out aminoacid replacement and do not influence the BA of proteins of interest matter is seen Dayhoff etc.; (1978); " protein sequence and structure iron " (Atlas of Protein Sequenceand Structure); (Natl.Biomed.Res.Found., Washington, model D.C.).Screening known in the art contains the combinatorial library gene product of passing through point mutation or brachymemma (method) preparation of selected characteristics and the method for screening cDNA library gene product.This method is applicable to that rapid screening passes through the gene library of the HVI labeling polypeptide that combinatorial mutagenesis produces.But coupling can improve the technology-recursion group mutagenesis (REM) of functional mutation frequency in this library identifies HVI labeling polypeptide variant (Arkin and Yourvan, (1992), Proc.Natl.Acad.Sci.USA, 89:7811-7815 with shaker test; Delgrave etc., (1993), Protein Engineering, 6:327-331).Hereinafter has detailed the ideal conservative property and has replaced, and for example has certain monoamino-acid of amino acid replacement of similar characteristics with another.

Compare with parental generation HVI marker amino acid sequence, variant HVI labeling polypeptide can contain conservative amino acid at the different positions along their sequences and replace." conservative amino acid replacement " is to replace certain amino-acid residue with the amino-acid residue with similar side chain.This area has defined the amino-acid residue family with similar side chain, and they generally can be further divided into:

Acid: this residue is electronegative because of under physiological pH, losing the H ion, and this residue is attracted by the aqueous solution, and therefore when the peptide that comprises it was in the physiological pH aqueous medium, this residue was in the surface location of this peptide configuration.The amino acid that contains acid side-chain comprises L-glutamic acid and aspartic acid.

Alkalescence: this residue is because of combining the H ion positively charged (for example under physiological pH or in its one or two pH unit; Histidine); This residue is attracted by the aqueous solution, and therefore when the peptide that comprises it was in the physiological pH aqueous medium, this residue was in the surface location of this peptide configuration.The amino acid that contains basic side chain comprises l-arginine, Methionin and Histidine.

Charged: these residues are electrically charged when physiological pH, therefore comprise the amino acid (that is, L-glutamic acid, aspartic acid, l-arginine, Methionin and Histidine) that contains acidity or basic side chain.

Hydrophobicity: these residues neutral and repelled by the aqueous solution under physiological pH, therefore when the peptide that comprises it was in the aqueous medium, this residue was in the interior location of this peptide configuration.The amino acid that contains hydrophobic side chain comprises tyrosine, Xie Ansuan, Isoleucine, leucine, methionine(Met), phenylalanine(Phe) and tryptophane.

Neutrality/polarity: these residues are neutral under physiological pH, but the aqueous solution is not enough to repel them, and therefore when the peptide that comprises this residue was in the aqueous medium, this residue was adsorbed in the interior location of this peptide configuration.The amino acid that contains neutrality/polar side chain comprises l-asparagine, Stimulina, halfcystine, Histidine, Serine and Threonine.

This specification sheets also is accredited as " little " amino acid with some amino acid characteristics, because their side chain big inadequately (even lacking polar group) thereby can not hydrophobic property.Except that proline(Pro), " little " amino acid is those amino acid that when at least one polar group is arranged on the side chain, have 4 following carbon and when not having polar group on the side chain, have 3 following carbon.Amino acid with little side chain comprises glycocoll, Serine, L-Ala and Threonine.The secondary amino acid of genes encoding--proline(Pro) is because of the influential special case that becomes of known its secondary configuration to peptide chain.The structure of proline(Pro) combines to be different from the amino acid of all other natural generations because of its side chain and alpha-amino nitrogen-atoms and alpha-carbon atom.For example Dayhoff etc. ((1978), " model that protein evolution changes " (A model of evolutionary change in proteins)) discloses a few seed amino acid similarity matrixs (for example, PAM120 matrix and PAM250 matrix).Yet; Publish in " protein sequence and structure iron " (Atlas of Protein Sequence andStructure) that M.O.Dayhoff compiles; The 5th volume; The 345-358 page or leaf, national biomedical research foundation (NationalBiomedical Research Foundation), Washington DC; With Gonnet etc., 1992, Science, 256 (5062): the matrix that is used for measuring long-range relation in 144301445 is included in proline(Pro) glycocoll, Serine, L-Ala and Threonine same group.Therefore, proline(Pro) is classified as " little " amino acid for the object of the invention.

(amino acid) is divided into polarity or required attraction or the repulsion degree of nonpolar classification is random, so the special amino acid of considering of the present invention can be divided into one type or another kind of.Can classify according to the amino acid that known performance is not named great majority specially.

Amino-acid residue also can be further divided into ring-type or non-annularity, aromatic series or non-aromatic, according to the classification of can justifying oneself of the side chain substituents of residue, be divided into little or big.If certain residue contains 4 following carbon atoms (comprising carboxyl carbon) altogether, think that it is a p1 amino acid, prerequisite is to have other polar substituent; If there is not other polar substituent, the residue that contains 3 following carbon atoms altogether can be thought p1 amino acid.Little residue is non-aromatic residue always certainly.Can it be divided into two or more kinds according to the structure properties of amino-acid residue.With regard to the gal4 amino acid of natural generation, see table 3 according to the subclassification of this scheme.

The conservative amino acid replacement also comprises according to side chain classifies.For example, the one group of amino acid that contains aliphatic lateral chain is glycocoll, L-Ala, Xie Ansuan, leucine and Isoleucine; The one group of amino acid that contains aliphatic series-hydroxyl side chain is Serine and Threonine; One group of amino acid with amide containing side chain is l-asparagine and Stimulina; The one group of amino acid that contains the aromatic series side chain is phenylalanine(Phe), tyrosine and tryptophane; The one group of amino acid that contains basic side chain is Methionin, l-arginine and Histidine; With one group of amino acid with sulfur-containing side chain be halfcystine and methionine(Met).For example; Can reasonably expect to replace with Isoleucine or Xie Ansuan that leucine, Stimulina replace l-asparagine, Serine replaces Threonine, can not have a strong impact on the performance of the variant polypeptide that is produced during perhaps with certain seed amino acid of amino acid similarity ground replacement of structurally associated.Whether amino acid change can produce functional HVI labeling polypeptide is not difficult through checking its activity to measure.Conservative property replaces the exemplary substituted title of seeing table 4.Preferred replacement title is seen in preferred replacement.Aminoacid replacement in the scope of the invention generally through selected substituting group to keeping: (a) replace regional peptide backbone structure, (b) electric charge of this molecule target site or hydrophobicity, or (c) volume of side chain does not have the replacement of obvious different effects to realize.After introducing replacement, screening has the variant of BA.

Perhaps, can will prepare the substituted similar amino acid of conservative property according to the homogeny of side chain is divided three classes.Like Zubay, G., " biological chemistry " (Biochemistry), the third edition, Wm.C.Brown Publishers, (1993) are said, first group comprises L-glutamic acid, aspartic acid, l-arginine, Methionin, the Histidine that all contains electrically charged side chain; Second group comprises glycocoll, Serine, Threonine, halfcystine, tyrosine, Stimulina, l-asparagine; The 3rd group comprises leucine, Isoleucine, Xie Ansuan, L-Ala, proline(Pro), phenylalanine(Phe), tryptophane, methionine(Met).

Therefore, replace the non-essential amino acid residue of estimating in the HVI labeling polypeptide with another amino-acid residue in the identical side chain family usually.Perhaps, can introduce sudden change at random along HVI marker gene encoding sequence all or part of, for example through saturation mutagenesis, the two mutants that obtains of screening has or not the activity of parental generation polypeptide to identify and has kept this active two mutants then.Behind the mutagenesis encoding sequence, can recombinant expressed coded peptide, measure the activity of this peptide.

Therefore, the present invention also considers variant or their biological active fragment of the HVI labeling polypeptide sequence of natural generation, and wherein said variant can be through adding, lack or replacing one or more amino-acid residues and distinguish mutually with the sequence of natural generation.Generally; Variant can show and parental generation HVI labeling polypeptide sequence that for example following SEQ ID NO:3, arbitrary sequence shown in 5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114 have the similarity at least about 30,40,50,55,60,65,70,75,80,85,90,91,92,93,94,95,96,97,98,99%.Variant preferably with parental generation HVI labeling polypeptide sequence, for example following SEQ ID NO:3, arbitrary sequence shown in 5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114 have the sequence homogeny at least about 30,40,50,55,60,65,70,75,80,85,90,91,92,93,94,95,96,97,98,99%.In addition, the present invention consider through add, lack or replace 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,30,40,50,60,70,80,90,100,150,200,300,500 with upper amino acid with natural or the parental generation sequence is different but still kept the sequence of this parental generation HVI labeling polypeptide characteristic.The HVI labeling polypeptide also comprises can be under rigorous condition defined herein, the polypeptide of the polynucleotide encoding of hybridizing with the invention described above HVI mark polynucleotide sequence or its noncoding strand under the particularly high rigorous condition.

In one embodiment, variant polypeptide has at least one with the HVI flag sequence but to be less than 50,40,30,20,15,10,8,6,5,4,3 or 2 amino-acid residues different.In another embodiment, variant polypeptide and following SEQ ID NO:3, the corresponding sequence shown in 5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114 have at least 1% but to be lower than 20%, 15%, 10% or 5% residue different.If (this compares (method) and needs comparison, then should compare the maximum comparability of these sequences.Because of disappearance or " ring " outer sequence that insertion or mispairing produced can think that difference is arranged).These difference preferably are positioned at the difference or the change of nonessential residue, or conservative property replaces.

" nonessential " amino-acid residue is can from the wild-type sequence of certain concrete polypeptide, change and do not destroy or do not change its residue a kind of or various active basically.Said change does not change basically preferably that these are one of active, and for example 20% of wild-type activity, 40%, 60%, 70% or 80%." essential " thus amino-acid residue is the activity that when changing this residue of HVI labeling polypeptide wild-type sequence of the present invention, can destroy this parental generation molecule is lower than the activity 20% of wild type peptide.

In other embodiments; Variant polypeptide comprises the corresponding sequence with the HVI labeling polypeptide, and for example following SEQ ID NO:3, arbitrary sequence shown in 5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114 have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%95%, 96%, 97%, 98% or above similarity and have the active aminoacid sequence of this HVI labeling polypeptide.

Can known by one of skill in the art any appropriate method prepare HVI labeling polypeptide of the present invention.For example, can prepare these polypeptide through the method that may further comprise the steps: (a) preparation contains the mosaic type construction of the nucleotide sequence of encoding certain HVI mark polynucleotide at least a portion and linking to each other with the regulatory element operability; (b) this mosaic type construction is introduced in the host cell; (c) cultivate this HVI labeling polypeptide of this host cell expression; (d) from host cell, separate this HVI labeling polypeptide.In exemplary example; Said nucleotide sequence coded following SEQ ID NO:3, sequence shown in 5,9,11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114, or at least a portion of its variant.

The mosaic type construction is taked the form of expression vector usually, should be selected from the outer carrier (for example plasmid) of karyomit(e) of self-replication and can be integrated into the carrier in the host genome.

Regulatory element generally should be suitable for expressing the used host cell of this HVI labeling polypeptide.The expression vector of many types of various host cells known in the art and regulatory element.The demonstrative component of this type includes but not limited to: promoter sequence (for example can be the composing type or the inducible promoters of natural generation or the composition element with multiple promotor), leading or signal sequence, ribosome bind site, transcription initiation and terminator sequence, translation initiation and terminator sequence and enhanser or activation sequence.

In some embodiments, expression vector comprises selectable marker gene to select transformed host cells.But selectable marker gene is well known in the art and different with used host cell.

Said expression vector also can comprise fusion partner (being provided by this expression vector usually) thereby can the HVI labeling polypeptide be prepared as the fusion polypeptide that contains fusion partner.The major advantage of fusion partner is that they help to identify and/or this fusion polypeptide of purifying.Be the preparation fusion polypeptide, need HVI mark polynucleotide be connected into fusion partner is overlapped with the translation frame of HVI mark polynucleotide.The well-known examples of fusion partner includes but not limited to: Fc part, maltose binding protein (MBP) and six Histidines (hexahistidine) (HIS of glutathione-S-transferase (GST), human IgG ₆), these companions are particularly useful for separating fusion polypeptide through affinity chromatography.In some embodiments, capable of using and fusion partner bonded matrix (such as but not limited to: gsh-, amylose starch-and nickel-or cobalt-coupling resin) through these fusion polypeptide of affinitive layer purification.Many this matrix can " test kit " form be buied, and for example utilize (HIS ₆) companion's QIAexpress ^TMSystem (Qiagen) and Pharmacia GST purification system.Other fusion partner known in the art is a photoprotein, for example can be used as fluorescence " label " identified and/or separated fusion polypeptide through fluorescent microscopy or flow cytometry green fluorescent protein (GFP) and luciferase.The flow cytometry method, for example fluorescence-activated cell sorting (FACS) method is particularly useful in use this back one.

Fusion partner also preferably contains proteolytic enzyme cutting site, for example factor X _aOr the cleavage site of zymoplasm, thereby make the GAP-associated protein GAP enzyme partly digest this fusion polypeptide and from fusion construct, discharge the HVI labeling polypeptide.Can separate polypeptide and the fusion partner that is discharged through chromatographic separating process subsequently.

The scope of fusion partner also comprises " epi-position label ", and these labels normally can be buied the short peptide sequence of its specific antibody.The well-known examples that is easy to buy the epi-position label of monoclonal antibody specific comprises c-Myc, influenza virus hemagglutinin and FLAG label.

Can mosaic type construction of the present invention be introduced among the host through comprising " transduction " and proper methods such as " transfections ", this means the transgenosis that mediates through nucleic acid technically with nucleic acid, and for example expression vector is introduced in the donee's cells.Yet the genotype that " conversion " refers to the host is taken in the process that exogenous DNA or RNA change because of cell, and for example transformant contains expression system of the present invention.The mosaic type construction is introduced cell many methods are arranged.Used method generally depends on the selection of host cell.Those skilled in the art know the technology of the mosaic type construction being introduced host cell.Described four types nucleic acid molecule sent the method into cell: (1) chemical process, the for example deposition and the lipofection of calcium phosphate precipitation, polyoxyethylene glycol (PEG)-mediation; (2) physical method, for example microinjection, electroporation, accelerated method and vacuum are infiltrated; (3) carrier method, the for example conversion of bacterium and virus vector mediation; (4) receptor-mediated conversion.Those skilled in the art know these and the transformation technology of other type, and new technology also continues to bring out.Concrete selection transformation technology depends on the efficient of this technical transform particular host cell and experience and the preference that adopts the people of the concrete method embodiment of the present invention of selecting.The technician can know that obviously the concrete selection with the conversion system in the mosaic type construction introducing cell is not substantial or limitation of the present invention, as long as this system can realize acceptable nucleic acid transfer level.

Can prepare reorganization HVI labeling polypeptide through cultivating through mosaic type construction transformed host cells.With host cell and different, the technician is through normal experiment definite these conditions of being not difficult with selected expression vector for condition that be fit to express the HVI labeling polypeptide.The host cell that is fit to express can be protokaryon or eukaryotic cell.The exemplary host cell of expressing polypeptide of the present invention is a bacterium.Used bacterium can be intestinal bacteria (Escherichia coli).Perhaps, host cell can be yeast cell or insect cell, for example utilizes the SF9 cell of baculovirus expression system.

Can adopt like Sambrook etc., (1989, the same), particularly the 16th and 17 parts; Ausubel etc., (1994, the same), particularly the 10th and 16 chapters; With Coligan etc.; " fresh approach of protein science " (CURRENT PROTOCOLS IN PROTEIN SCIENCE), (John Wiley&Sons, Inc.; 1995-1997), particularly the standard method described in the 1st, 5 and 6 chapters is not difficult to prepare reorganization HVI labeling polypeptide.Perhaps, can be through chemosynthesis, for example adopt like Atherton and Shephard, (the same) the 9th Zhanghe Roberge etc., (1995, Science, the liquid phase described in 269:202) is synthesized or solid-phase synthesis comes chemosynthesis HVI labeling polypeptide.

4. antigen binding molecules

The present invention also provide can with the interactional antigen binding molecules of HVI labeling polypeptide generation specific immunity of the present invention.In one embodiment, this antigen binding molecules comprises the polyclone whole antibody.Can pass through, for example HVI labeling polypeptide of the present invention is injected into the animal (comprising mouse and rabbit) that can produce (antibody) and prepare this antibody to obtain polyclonal antiserum.Those skilled in the art know the method for preparing polyclonal antibody.Adoptable exemplary method sees, Coligan etc. for example, " immunology fresh approach " (CURRENTPROTOCOLS IN IMMUNOLOGY); (John Wiley&Sons, Inc, 1991) and Ausubel etc.; (1994-1998, the same), particularly the III part of Chapter 11.

Can adopt; For example K

hler and Milstein (1975, Nature, 256; 495-497) described standard method or employing; Coligan etc. for example, (1991, the same) described up-to-date improving one's methods; Infinite multiplication splenocyte or other antibody produced cell with deriving from generation (antibody) animal that has inoculated one or more HVI labeling polypeptides of the present invention prepare monoclonal antibody, substitute from producing the polyclonal antiserum that (antibody) animal obtains.

The present invention also considers with Fv, Fab, Fab ' and F (ab ') ₂Immunoglobulin fragment is as antigen binding molecules.Perhaps, antigen binding molecules can comprise the Fv fragment that synthetic is stable.The exemplary fragment of this type comprises with peptide linker difference bridging V _HThe N-terminal of structural domain or C-terminal and V _LThe strand Fv fragment (sFv is commonly referred to scFv) that the C-terminal of structural domain or N-are terminal.All constant regions of ScFv lack of complete antibody, thereby can not activating complement.Can basis, for example Kreber etc. (Kreber etc., 1997, J.Immunol.Methods, 201 (1): the method for 35-55) being summarized prepares ScFv.Perhaps, can pass through U.S. Patent number 5,091,513; European patent number 239; 400 or the article (1991 of Winter and Milstein; Nature; 349:293) with Pl ü ckthun etc. (1996, publish in " antibody engineering: a kind of practical approach " (Antibodyengineering:Apractical approach), 203-252) described method prepares them.In another embodiment, the stable Fv fragment of synthetic comprises the stable Fv of disulfide linkage (dsFv), in this fragment, two cysteine residues is introduced V _HAnd V _LThereby in the structural domain in folding fully molecule this two residue form disulfide linkage to each other.The appropriate method that produces dsFv is described in, for example (Glockscuther etc., Biochem., 29:1363-1367; Reiter etc., 1994, J.Biol.Chem., 269:18327-18331; Reiter etc., 1994, Biochem., 33:5451-5459; Reiter etc., 1994, CancerRes., 54:2714-2718; Webber etc., 1995, Mol.Immunol., 32:249-258).

Preparation known in the art is anti--and the phage display of the antigen binding molecules of HVI labeling polypeptide and combined method be (for example, described in the following document: Ladner etc., U.S. Patent number 5,223,409; Kang etc., international publication number WO92/18619; Dower etc., international publication number WO91/17271; Winter etc., international publication WO92/20791; Markland etc., international publication number WO92/15679; Breitling etc., international publication WO93/01288; McCafferty etc., international publication number WO92/01047; Garrard etc., international publication number WO92/09690; Ladner etc., international publication number WO90/02809; Fuchs etc., (1991), Bio/Technology, 9: 1370-1372; Hay etc., (1992), Hum Antibod Hybridomas, 3: 81-85; Huse etc., (1989), Science, 246: 1275-1281; Griffths etc., (1993), EMBOJ, 12: 725-734; Hawkins etc., (1992), JMolBiol, 226: 889-896; Clackson etc., (1991), Nature, 352: 624-628; Gram etc., (1992), PNAS, 89: 3576-3580; Garrad etc., (1991), Bio/Technology, 9: 1373-1377; Hoogenboom etc., (1991), Nuc AcidRes, 19: 4133-4137; With Barbas etc., (1991), PNAS, 88: 7978-7982).These antigen binding molecules can be used for screening the variant HVI labeling polypeptide in the expression library.They also can be used for detecting and/or separating HVI labeling polypeptide of the present invention.Therefore, the present invention also considers to utilize these antigen binding molecules, adopts for example any suitable immune affine method (including but not limited to immunochromatography and immunoprecipitation) to separate the HVI labeling polypeptide.Suitable method is utilized solid phase adsorption, wherein will resist-antigen binding molecules of HVI labeling polypeptide links to each other with appropriate resin, and this resin contact with suspecting the sample that contains the HVI labeling polypeptide, then wash-out HVI labeling polypeptide (if existence) from the resin.Exemplary resin comprises: Sepharose ^TM(Pharmacia), Poros ^TMResin (Roche Molecular Biochemicals, Indianapolis), ActigelSuperflow ^TMResin (Sterogene Bioseparations Inc., Carlsbad Calif.) and Dynabeads ^TM(Dynal Inc., Lake Success, N.Y.).

Can be with antigen binding molecules and compound, affinity tag for example, like radionuclide, or imaging agents, like radioactivity, enzymic activity (reagent) or other imaging agents, like the coupling of NMR contrast agent phase.The preferred affinity tag that can produce detectable radioactive ray or fluorescence.Be abundance and pattern that evaluating protein matter is expressed, capable of using anti--antigen binding molecules (for example, monoclonal antibody) of HVI labeling polypeptide detects HVI labeling polypeptide (for example, in cell lysate or cell conditioned medium liquid).In some advantageous applications of the present invention, available this antigen binding molecules comes the HVI labeling polypeptide level in the monitoring biological sample (containing complete cell and liquid) and diagnoses the risk that whether has herpesvirus infection and degree or herpesvirus infection associated diseases.Antibody and detectable substance (that is antibody labeling) phase coupling (that is physical connection) are helped to detect.The example of detectable substance comprises various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material and radio active material.The example of suitable enzyme comprises horseradish peroxidase, SEAP, beta-galactosidase enzymes or E.C. 3.1.1.7; The example of suitable prothetic group mixture comprises Streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent material comprises Umbelliferone, resorcinolphthalein, fluorescein isothiocyanate, rhodamine, dichloro three azine amine resorcinolphthaleins (dichlorotriazinylamine fluorescein), dansyl chloride or phycoerythrin; The example of luminescent material comprises o-aminophthalylhydrazide; The example of bioluminescent material comprises luciferase, resorcinolphthalein and aequorin; The example of suitable radio active material comprises 125 _I, 131 _I, 35 _SOr 3 _HAffinity tag can be selected from chromogen, catalyzer, enzyme, fluorophor, chemiluminescent molecule, lanthanide ion, like europium (Eu ³⁴But), the affinity tag of ri and direct viewing.But with regard to the direct viewing affinity tag, available colloidal metal particle or non-metallic particle, dye granule, enzyme or substrate, organic polymer, latex particle, liposome or contain the preparation marks such as other carrier that signal produces material.

US Patent specification U.S.4,366,241; U.S.4,843,000 and U.S.4,849,338 disclose the enzyme that can be used as affinity tag in a large number.Available enzyme labelling thing of the present invention comprises SEAP, horseradish peroxidase, luciferase, beta-galactosidase enzymes, P-FAD, N,O-Diacetylmuramidase, MDH etc.Can use separately enzyme labelling thing solution or with second kind of enzyme coupling.

5. detecting unusual HVI marker gene expresses or allelic method

The present invention is partly based on following discovery: compare with the horses that do not receive herpesvirus infection; Receive some gene of horses, particularly those horses of reactivity herpesvirus infection that simplexvirus (particularly EHV) infects or some allelotrope (this paper is called the HVI marker gene) abnormal expression of these genes.This paper proposes in receiving other animal of herpesvirus infection, to find these genes or their homologue or straight abnormal expression to homologue.Therefore; The invention is characterized in that the unconventionality expression of taking from certain HVI marker gene in the biological sample of object through detection assesses herpesvirus infection or the diagnosis herpesvirus infection, the particularly method of reactivity herpesvirus infection of this object (preferred mammal).Herpesvirus infection should comprise that one of simplexvirus subfamily member of being selected from α herpetoviridae, β herpetoviridae and gamma herpes viruses section infects.The exemplary of the simplexvirus of α herpetoviridae subfamily comprises: HHV-1 (HSV-1), HHV-2 (HSV-2), HHV-3 (VZV) and EHV (as, EHV-1 and EHV-4).The non-limitative example of the simplexvirus of β herpetoviridae subfamily comprises: HHV-5 (CMV), HHV-6 (roseola virus) and HHV-7.The representative example of the simplexvirus of gamma herpes viruses section subfamily includes but not limited to: and HHV-4 (the lymph cryptovirus, EBV) and HHV-8 (elongated virus).In specific embodiment, said simplexvirus is EHV, and particularly EHV-1 is more especially reactivity EHV-1.

For making assessment or diagnosis, need qualitatively or quantitatively determine the level of HVI marker gene transcript, the specific allelic existence of certain HVI marker gene or the level or the functionally active of level or HVI labeling polypeptide.In some embodiments; Certain the HVI marker gene product detection level that in biological sample, exists is lower than to be taken from normal subjects or not to receive herpesvirus infection; When particularly not receiving the simplexvirus reactivity to infect the level of this gene in the reference sample of object; Diagnosable herpesvirus infection state, degree or stage, perhaps the existence generation simplexvirus sequela risk of existing.In other embodiments; But the detection level of certain HVI marker gene product is higher than and takes from normal subjects or do not receive herpesvirus infection in biological sample; When particularly not receiving the simplexvirus reactivity to infect the level of this gene of reference sample of object; Then diagnosable herpesvirus infection state, degree or stage, perhaps the existence generation simplexvirus sequela risk of existing.In a word; The level of corresponding HVI marker gene product or functionally active are compared and are had 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99% at least in the level of certain the HVI marker gene product in the biological sample or functionally active and reference sample; Or even at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%; Or even during at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% difference, can make this diagnosis.Table 5-7 is seen in the exemplary rising of representative HVI marker gene expression level or reduction.

Corresponding gene product is selected from the same gene product that exists in the biological sample, variant gene (for example, homology or orthologous gene) expressed genes product or their protein that contains allele variant or splice variant usually.In some embodiments, this method comprises that detection is at least about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or the level or the functionally active of 30 kind of each expression product of HVI marker gene.

Biological sample generally comprises blood, peripheral blood particularly, or its component or extract.Biological sample contains hemocyte usually; For example ripe, immature and grow white corpuscle; Comprise lymphocyte, polymorphonuclear leukocyte, neutrophil, monocyte, reticulocyte, basophilic granulocyte, chamber born of the same parents, hemocyte, eosinophilic granulocyte, megalokaryocyte, scavenger cell, dendritic cell, natural killer cell; Or the component of these cells (for example, nucleic acid or protein portion).In specific embodiment, biological sample contains white corpuscle, comprises PMBC (PBMC).

5.1 Diagnostic nucleic acid

Can according to standard method (Sambrook etc., 1989, the same; Ausubel etc., 1994, the same) isolating nucleic acid is used for the polynucleotide test from the contained cell of biological sample.Said nucleic acid generally is fractionated (for example, poly A ⁺RNA) or whole-cell rna.When during as detected object, need RNA being changed RNA into complementary DNA.In some embodiments, adopt this nucleic acid of template dependency nucleic acid amplification technologies amplification.Existing many template dependency methods increase, and certain gives the HVI flag sequence that exists in the solid plate sample.Exemplary nucleic acid amplification technologies is specified in U.S. Patent number 4,683,195; 4,683,202 and 4,800,159; Polymerase chain reaction (being called PCR) among Ausubel etc. (the same) and the Innis etc., (" PCR method " (PCRProtocols), Academic Press, Inc., San Diego Calif., 1990).In brief, in PCR, two primer sequences of regional complementarity on the relative complementary strand of preparation and this flag sequence.In reaction mixture, add excessive deoxynucleoside triphosphate and archaeal dna polymerase, for example Taq polysaccharase.If there is homology HVI flag sequence in certain sample, these primers will combine with this mark (sequence), and polysaccharase can make this primer extend along flag sequence through adding Nucleotide.Through raising and reduce the temperature of reaction mixture, the primer of extension can with mark (sequence) the formation reaction product of dissociating, excessive primer can combine with mark (sequence) and reaction product, and carries out this process repeatedly.MRNA content for quantitatively determined is increased can adopt the reverse transcriptase PCR amplification method.With the RNA rt is that the method for cDNA is known, and it is described in Sambrook etc., 1989, and the same.Other rt method is utilized heat-staple RNA dependent dna-polymerases.WO90/07641 has described these methods.Well known polymerase chain reaction method.

In some preferred embodiment, the template dependent amplification comprises real-time quantitative mensuration transcript.For example, can adopt real time pcr (Higuchi etc., 1992, Biotechnology, 10:413-417) quantitatively determined RNA or DNA.Through measuring the concentration of accomplishing similar number amplification round in the PCR reaction and being in the target DNA amplified production in the linearity range, can confirm the relative concentration of specific target sequence in the original DNA mixture.If the DNA mixture is the relative abundance from the specific mrna that separates from the RNA of different tissues or cell synthetic cDNA, then can measure each tissue that produces this target sequence or cell.This direct proportional relationship between the concentration of PCR product and the mRNA relative abundance is only just correct in the linearity range of PCR reaction.Can measure the final concentration of this curve terrace part target DNA through the utilization ratio of reagent in the reaction mixture, this final concentration does not rely on the starting point concentration of this target DNA.

Another amplification method is the disclosed ligase chain reaction LCR of EPO No.320308 (" LCR ").In LCR, prepare two pairs of complementary probe, thereby the opposed complementary chain combination of the every pair of probe and target sequence is adjoined them in the presence of target sequence.In the presence of ligase enzyme, these two pairs of probes can link to each other and form a unit.With the same among the PCR, through temperature cycle, make bonded connect unit and dissociate, then with connecting excess probe right " target sequence " from target sequence.U.S. Patent number 4,883,750 described similar with LCR, make probe to target sequence bonded method.

Also can use the described Q β of PCT application number PCT/US87/00880 replicative enzyme.In this method, in the presence of RNA polymerase, will have with the rna replicon sequence of the regional complementary region mutually of target sequence and add in the sample.Polysaccharase can copy this replication sequence, detects then.

Utilize the amplification of restriction endonuclease and ligase enzyme also to can be used for the nucleic acid of the present invention that increases in the constant-temperature amplification method that the restriction site of a chain contains the target molecule of Nucleotide 5 ' α-sulfo--triphosphoric acid; Walker etc.; (1992; Proc.Natl.Acad.Sci.U.S.A, 89:392-396).

Strand displacement amplification (SDA) is the another kind of method of carrying out nucleic acid constant-temperature amplification, and this method comprises many wheel strand displacements with synthetic, i.e. nick translation.The similarity method that is called reparation Kettenreaktion (RCR) comprises anneals several kinds of probes on the entire target zone of amplification, only repair reaction to two kinds in four kinds of bases then.For ease of detecting other two kinds of bases are added as the biotinylation verivate.Adopted similar method among the SDA.Also can adopt circle probe reaction (CPR) detection specificity target sequence.In CPR, has the DNA hybridization that exists in probe and the sample of 3 ' and 5 ' sequence and specific RNA stage casing sequence of non-specific DNA.After the hybridization, with RNA enzyme H processing reaction (mixture), the product of probe is accredited as the different products that the digestion back discharges.Make the annealing of primary template and another circle probe, repeat this reaction.

Also can adopt GB Patent Application No. 2202328 and the described another kind of amplification method of PCT application number PCT/US89/01025.In last application, the template and the enzyme dependency that " modification " primer are used for PCR-appearance are synthetic.Available part (for example, vitamin H) and/or these primers of test section (for example, enzyme) mark modification of catching.In one application of back, excessive label probe is added sample.In the presence of target sequence, probe combines and is cut by enzyme catalysis.After the cutting, the complete release of target sequence and then combine with excess probe.The cutting of label probe is the signal that target sequence exists.

Other nucleic acid amplification method comprises transcription amplification system (TAS), comprises amplification of nucleic acid sequences (NASBA) and 3SR (Kwoh etc., 1989, Proc.Natl.Acad.Sci.U.S.A., 86:1173; Gingeras etc., PCT applies for WO88/10315).In NASBA, can the extraction of standard phenol/chloroform, thermally denature, lysis buffer through clinical sample handle and microcentrifugation (minispin) post DNA isolation prepares the nucleic acid that is used to increase with RNA or through chlorination guanidine extraction RNA.These amplification techniques comprise makes the primer annealing with target-specific sequence.After the polymerization, the DNA/RNA crossbred with RNA enzyme H digestion hybridization carries out thermally denature again to double chain DNA molecule.In two kinds of situation, thus through add second target-specific primer then polymerization single stranded DNA is processed complete two strands.Use RNA polymerase then, for example T7 or SP6 transcribe this double chain DNA molecule for many times.In the constant temperature circulating reaction, be single stranded DNA with the RNA rt, change double-stranded DNA then into, then use RNA polymerase, for example T7 or SP6 transcribe again.No matter the product that obtains is brachymemma or the complete target-specific sequence that all is shown as.

Davey etc., EPO No.329 822 discloses a kind of nucleic acid amplification method, comprises can be used for circulation synthetic single stranded RNA of the present invention (" ssRNA "), ssDNA and double-stranded DNA (dsDNA).SsRNA is the template of first primer tasteless nucleotide of reversed transcriptive enzyme (RNA dependent dna-polymerases) extension.Utilize the effect of ribonuclease H (RNA enzyme H has specific RNA enzyme to RNA in the duplex that contains DNA or RNA) to remove the RNA in the DNA:RNA duplex that obtains then.The ssDNA that produces is the template of second primer, and this primer also contains the promoter sequence with this template homologous RNA polymerase (for example t7 rna polymerase) at 5 ' (end).Use archaeal dna polymerase (for example big " Klenow " fragment of e. coli dna polymerase I) to extend this primer then, obtain double-stranded DNA (" dsDNA ") molecule, its sequence is identical with original RNA between the primer, also contains promoter sequence at an end.Suitable R NA polysaccharase can utilize the many parts of RNA copies of this DNA of this primer sequence preparation.Thereby these copies can get into the circulation rapid amplifying more then.Suitably select enzyme, but constant temperature carries out this amplification (method) and need not at every adding enzyme of taking turns.Owing to the recursive nature of this method, can select the homing sequence of DNA or rna form.

Disclosed amplification of nucleic acid sequences scheme in PCT application WO89/06700 such as Miller is transcribed many parts of RNA copies of this sequence then according to promotor/primer sequence and strand target DNA (" ssDNA ") hybridization.This scheme is not circulative, and the rna transcription thing that promptly obtains does not produce new template.Other amplification method comprises " RACE " and " monolateral (one-sided) PCR " (Frohman; M.A., publish in " PCR method: method and application guide " (PCR Protocols:A Guide to Methods andApplications), Science Press; N.Y., 1990; Ohara etc., 1989, Proc.NatlAcad.Sci.U.S.A., 86:5673-567).

Also can be employed in the nucleic acid that contains to some extent " two-oligonucleotide " sequence that produces exist connect two (or many) down thus the method that the oligonucleotide amplification should " two-oligonucleotide " is come amplifying target nucleic acid sequence.Wu etc., (1989, Genomics, 4:560).

Depend on used mode, can adopt for example above-mentioned template dependent amplification or after amplification, directly identify interested HVI labeling nucleic acid in the sample with second kind of known nucleic acid.Detect the product of being identified then.In some applications, can detect (for example, gel ethidium bromide staining) through visual method.Perhaps, detection can comprise through chemoluminescence, radiolabeled radioactivity scintiphotography or fluorescent marker or even system through utilizing electricity or thermal pulse signal to identify indirectly product (Affymax Technology; Bellus, 1994, JMacromol.Sci.Pure, Appl.Chem., A31 (1): 1355-1376).

In some embodiments, can observe amplified production or " amplicon " to confirm the amplification of HVI flag sequence.A kind of typical visual method comprises uses the ethidium bromide staining gel, under UV light, observes.Perhaps, if amplified production has been integrated mark radioactivity or fluorescently-labeled Nucleotide can make these amplified productions exposure x-radiographic films or observation under suitable excitation spectrum after the separation.In some embodiments, can observe indirectly.Amplified production makes the nucleic probe of mark contact with the HVI flag sequence of amplification after separating.Probe preferably with chromophore's coupling, but but also radio-labeling.Perhaps, with probe and binding partners, for example antigen binding molecules or vitamin H coupling, but and combine another right member to carry test section or report molecule.Related technology is well known to those skilled in the art, can in the standard textbook of many molecular methods (for example, referring to Sambrook etc., 1989, the same and Ausubel etc., 1994, the same), find.For example, can be during increasing or thereafter with chromophore or radiolabeled probe or primer evaluation target (sequence).

In certain embodiments, adopt engram technology quantitatively determined target nucleic acid well known to those skilled in the art.The Sourthern trace utilizes DNA as target, and the Northern trace utilizes RNA as target.Each method can provide different kinds of information, though the cDNA trace is similar with above-mentioned trace or RNA in many aspects.In brief, probe is used for target and is fixed in suitable matrix, normally DNA on the nitrocellulose filter or RNA.Different sorts nucleic acid spatially should separate so that analyze.Often through the nucleic acid gel electrophoresis, " trace " realized to film then for this.Subsequently, common trace target (sequence) and the probe (the normally probe of mark) cultivated under the condition that promotes sex change and hybridization (rehybridisation) again.Because probe design is and target (sequence) base pairing that probe can combine with the part of target sequence under the renaturation condition.Remove unconjugated probe then, detect as stated.

After detected/quantified is measured, can or not have herpesvirus infection, particularly do not have the reference group that statistical significance is arranged of reactivity herpesvirus infection object and make comparisons finding result and the control reaction (result) or the normal subjects of certain given object.Detected HVI labeling nucleic acid content is associated with the process or the seriousness of disease.

The present invention also considers like Kristensen etc.; (Biotechniques; 30 (2): 318-322) described methods of genotyping and allelotrope discrimination method and technology, these methods and technology comprise the single nucleotide polymorphisms analysis of employing, high performance liquid chromatography, TaqMan ^TM, LC and mass spectrum.

The present invention also considers biochip technology, for example Hacia etc. (1996, Nature Genetics, 14:441-447) with Shoemaker etc. (1996, Nature Genetics, 14:450-456) described technology.In brief, these technology comprise the method for quantitatively determining of the many genes of quick Accurate Analysis.Through giving the gene tagging with oligonucleotide or utilizing fixed probe array, available biochip technology that target molecule is separated into high density arrays and according to these molecules of screening by hybridization.Also referring to Pease etc. (1994, Proc.Natl.Acad.Sci.U.S.A., 91:5022-5026); Fodor etc. (1991, Science, 251:767-773).In brief, such as this paper general introduction, the nucleic probe of preparation HVI mark polynucleotide links to each other it with being used to screen with the biochip of diagnostic method.Can the nucleic probe that connect biochip be designed to basically and specific expressed HVI labeling nucleic acid; Be no matter target sequence (is target sequence or other probe sequence of sample; The sequence in the sandwich assay for example) complementation, thus target sequence and probe hybridization of the present invention made.This complementarity need not perfection; The base-pair mismatch that can have the interference hybridization of some amount between target sequence and the nucleic probe of the present invention.Yet if do not hybridize under minimum preciseness hybridization conditions even the quantity of mispairing is too big, this sequence is not the complementary sequence of target sequence yet.In certain embodiments, every sequence multiple probe capable of using, eclipsed probe capable of using or to the probe of target sequence different piece.That is, available two kinds, three kinds, the probe more than four kinds (preferred three kinds) make up concrete target (sequence) and have Feng Yudu.These probes can be eclipsed (being that some sequence is identical) or isolating.

Those of ordinary skills should be understood that and can in every way nucleic acid be connected in or be fixed in solid support.Being connected or being combined under following combination, washing, analysis and the removal condition enough stable between " fixed " of this paper or its grammer construed nucleic probe of equal value and the solid support.In conjunction with can being covalently or non-covalently.One or more of " the non-covalent combination " of this paper and its grammer construed static of equal value, wetting ability and hydrophobic interaction.Non-covalent combination comprises molecule, and for example Streptavidin combines with the non-covalent of Streptavidin with the covalently bound and biotinylated probe of upholder." covalent attachment " of this paper and grammer thereof construed two portions of equal value, promptly solid support and probe through a key, comprise that σ key, π key link to each other with co-ordination bond at least.Can be formed directly in covalent linkage between probe and the solid support, perhaps can form covalent linkage through cross linker or through solid support or probe the two or the contained specific reaction reactive group of these two kinds of molecules.Immobilization also can comprise and uniting with covalency and noncovalent interaction.

In a word, those skilled in the art will know that linking probe and biochip in every way.As described herein, can first nucleic acid, link to each other with biochip then, perhaps direct nucleic acid on biochip.

Biochip comprises suitable solid or semi-solid substrate or solid support." substrate " or " solid support " expression contains through modifying and is fit to link to each other with nucleic probe or discontinuous a plurality of sites of bonded and be applicable to any material of at least a detection method.Those skilled in the art will know that the available substrate is a lot, include but not limited to: glass and modification or functional glass, plastics (multipolymer of vinylformic acid, PS and vinylbenzene and other material, Vestolen PP 7052, Vilaterm, polybutylenes, urethane, Teflon ^TMDeng), polysaccharide, nylon or nitrocellulose, resin, silicon-dioxide or material (silicon that comprises modification), carbon, metal, unorganic glass, plastics etc.In a word, these substrates can detect and not be with fluorescence with optical means.

Said substrate is generally put down, though those skilled in the art will know that the substrate that also can utilize other configuration.For example, reduce sample volume as far as possible, can probe be placed the internal surface of test tube for the sample that flows through is analyzed.Similarly, said substrate can be flexible, and for example the bendable foam plastics comprise the porous plastics that the sealing cell is arranged that particular plastic is processed.

In certain embodiments, known in the art can be on substrate synthetic oligonucleotide probe.For example, utilize the photoactivation technology of photopolymerization compounds and technology.In an exemplary, adopt the light version printing technology of knowing, for example WO95/25116; WO95/35505; U.S. Patent number 5,700,637 and 5,445,934 and the reference quoted described in technological original position nucleic acid; These methods of attachment constitute Affymetrix GeneChip ^TMThe basis of technology.

In the example of an exemplary analyzing biochips, the oligonucleotide probe on the biochip is contacted at the nucleic acid samples that helps under the condition of specific hybrid to contain one or more simplexvirus polynucleotide with suspection.DNA or the RNA extract (no matter strand or two strands) that can from the suspension of biomaterial, prepare sample; Perhaps grind biomaterial; Perhaps according to the preparation of lysis step, said step includes but not limited to: handle with SDS (or other washing composition), osmotic pressure impact, guanidinium isothiocyanate and N,O-Diacetylmuramidase and carry out cracking.The suitable DNA that can be used for the inventive method comprises cDNA.Can adopt this DNA of one of multiple domestic method preparation, for example see Ausubel etc., 1994, the same; With Sambrook etc., 1989, with the above.

The suitable RNA that can be used for the inventive method comprises complementary RNA (cRNA) or geneome RNA or the subgenomic RNA that messenger RNA(mRNA), DNA transcribe.Can adopt standard method to prepare this RNA, Ausubel etc. for example, 1994, the same; With Sambrook etc., 1989, with the above.

Can pass through, for example ultrasonication or handle with restriction endonuclease and to make the cDNA fragmentation.Thereby it is preferably make the cDNA fragmentation make the dna fragmentation of generation be longer than the fixed oligonucleotide probe, but enough little and can under suitable hybridization conditions, contact the fixed probe fast.Perhaps, can adopt the for example above-mentioned suitable amplification oligonucleotide technology cDNA fragment of selecting and increase, this relate to utilize suitable at random or Auele Specific Primer.

Usually thereby detectability labels targets HVI mark polynucleotide can be measured the hybridization of they and each probe.Usually with report molecule detectability labels targets polynucleotide, the exemplary of said report molecule comprises chromogen, catalyzer, enzyme, optical dye, chemiluminescent molecule, bioluminescent molecules, lanthanide ion (for example, Eu ³⁴But), the affinity tag of ri and direct viewing.But for the affinity tag of direct viewing, colloidal metal particle capable of using or non-metallic particle, dye granule, enzyme or substrate, organic polymer, latex particle, liposome or contain other carrier etc. that signal produces material.This type of exemplary affinity tag comprises big colloid, and metallic colloid for example is like gold, selenium, silver, tin and titanium oxide colloid.At some but enzyme is used as in the embodiment of direct viewing affinity tag, biotinylated base is mixed in the target polynucleotide.Detect hybridization through cultivating with Streptavidin-report molecule.

Suitable optical dye includes but not limited to: fluorescein isothiocyanate (FITC), isothiocyanic acid tetramethyl-rhodamine (TRITC), R-phycoerythrin (RPE) and Texas red (Texas Red).Other exemplary optical dye comprises described in (international publication WO93/06121) such as Dower.Optical dye also can be with reference to USP 5,573,909 (Singer etc.); 5,326,692 (Brinkley etc.).Perhaps, optical dye can be with reference to U.S. Patent number 5,227,487; 5,274,113; 5,405,975; 5,433,896; 5,442,045; 5,451,663; 5,453,517; 5,459,276; 5,516,864; 5,648,270 and 5,723, described in 218.The fluorescent marker that can buy comprises, resorcinolphthalein phosphoramidite for example is like Fluoreprime ^TM(Pharmacia), Fluoredite ^TM(Millipore) and FAM (Applied Biosystems International).

Radioactivity report molecule comprises, for example can be through X-ray or phosphoric acid imaging (phosphoimager) technology for detection ³²P.

Can under the condition that is suitable for oligonucleotide probe and test nucleic acid (comprising DNA or RNA) hybridization, hybridize the formation step.Thus, but reference example like " nucleic acid hybridization, a kind of practical approach " (NUCLEICACID HYBRIDIZATION, A PRACTICAL APPROACH), (Homes compile with Higgins), (IRL press, Washington D.C., 1985).In a word, hybridization whether receive concentration, the hybridization formation of length, pH, temperature, monovalence and the divalent cation of oligonucleotide probe and the polynucleotide sequence of test distinguish in ratio, dielectric viscosity and the influence of the denaturing agent that possibly exist of G and C Nucleotide.These variable factors also influence the hybridization required time.Therefore, preferred condition depends on concrete application.Yet available ordinary method is confirmed these empirical conditions and need not too much experiment.

Some preferred embodiment adopts height distinctive hybridization conditions.For example; Can with reference to Wallace etc. (1979, Nucl.Acids Res., 6:3543); He has described with the similar oligonucleotide probe that contains an inner base-pair mismatch and has compared, and can distinguish and the hybridization conditions of target sequence Perfect Matchings with the oligonucleotide probe of complete homologous 11-17 base length.Also can be with reference to Wood etc. (1985; Proc.Natl.Acid.Sci.USA, 82:1585), he has described the condition of utilizing the 3M tetramethylammonium chloride to make the long oligonucleotide hybridization of 11-20 base; Wherein the melting temperature(Tm) of crossbred only depends on the length of oligonucleotide probe, and its GC content no matter.In addition, Drmanac etc. (the same) have described the rigorous hybridization conditions of the long oligomer of 6-10 Nucleotide, utilize nucleotide analog, for example " locked nucleic acid " be not difficult to obtain simulated condition (Christensen etc., 2001, Biochem J, 354:481-4).

Hybridization generally can carry out in the presence of hybridization buffer, optional hybridization optimization reagent, for example stablizer (isostabilising agent), denaturing agent and/or the renaturation accelerator of containing of said damping fluid.The example of stablizer includes but not limited to: trimethyl-glycine and rudimentary tetraalkylammonium salt.Denaturing agent is the compsn that reduces the double chain acid molecule melting temperature(Tm) through the hydration of hydrogen bond between the interference double-strandednucleic acid base or nucleic acid molecule.Denaturing agent includes but not limited to: methane amide, formaldehyde, DMSO 99.8MIN., tetraethyl-acetic ester (tetraethylacetate), urea, guanidinium isothiocyanate (guanidium isothiocyanate), glycerine and chaotropic salt.The hybridization accelerator comprises heterogeneous nuclear ribonucleoprotein (hnRP) A1 and cationic detergent in the nuclear, like cetyl trimethylammonium bromide (CTAB) and Trimethyllaurylammonium bromide (DTAB), polylysine, spermine, spermidine, single strand binding protein (SSB), phage T4 gene 32 albumen and ammonium acetate and alcoholic acid mixture.Hybridization buffer can contain concentration and be the about 50nM of about 0.005-, preferably about 0.5-5nM, the target polynucleotide of 1-2nM more preferably from about.

The hybridization mixture that contains target HVI mark polynucleotide is contacted with probe array and cultivate appropriate time so that the target sequence in the target polynucleotide is hybridized with any complementary probe in room temperature.Can in any suitable containers, contact, for example be designed for the dish or the cell that hold the solid support that is connected with probe on it.Cultivate temperature and be generally the used temperature of nucleic acid hybridization, for example about 20 ℃-Yue 75 ℃, 25 ℃ according to appointment, about 30 ℃, about 35 ℃, about 40 ℃, about 45 ℃, about 50 ℃, about 55 ℃, about 60 ℃, or about 65 ℃.Length surpasses the preferred 20-50 of probe ℃ of 14 Nucleotide.As far as short probe, preferred lesser temps.Target polynucleotide sample and probe cultivated when enough reach required hybridization level between the target sequence in the chien shih target polynucleotide and any complementary probe.For example, can in methane amide, hybridize 1-2 days at about 45 ℃+/-10 ℃.

After hybrid forms step, with contain optimize agent with the hybridization of the used same concentrations of hybridization step hybridization buffer washing probe to remove any unconjugated nucleic acid.This washing step only stays the bonded target polynucleotide.But detection probes is to identify the hybridization of which kind of probe and target polynucleotide then.

Can detect hybridization then to confirm which kind of probe and corresponding target sequence hybridization.The character that depends on the report molecule that is connected with target polynucleotide can be used the instrument detecting signal: use the rayed fluorescent marker, detect fluorescence with photofluorometer in the following manner; Provide enzyme system to produce the color that available spectrophotometer detects; Perhaps detect dye granule or colored colloidal metal particle or non-metallic particle with the light reflectometer; Adopt radiation counter or radioautography during with radioactively labelled substance or chemiluminescent molecule.Therefore, proofing unit should be applicable to detection or the relevant light of passing marker thing, and said light can comprise fluorescence, cold light, focused beam or laser.At this moment, charge available coupled apparatus (CCD) or sensitive cell scan each position probe in the microarray: the light of target polynucleotide hybrid emission is also directly at the digital machine identifying recording layer.In some cases, need not the detected electronically signal.For example, producing the color spot relevant with the nucleic acid array pattern with enzymatic is example, estimates this array with regard to the pattern on the soluble array.In the nucleic acid array situation, proofing unit preferably links to each other with mode identificating software and the signal mode of array is converted into ordinary language heredity distribution pattern.In certain embodiments, the specific oligonucleotide probe that different HVI stress the marker gene product is taked the nucleic acid array form, with the report signal that molecule produced on " chip-reader " detection arrays.(U.S. Patent number 5,143,854) such as detection system that " chip-reader " adopts such as Pirrung are said.Chip-reader comprises also that usually some signal courses of processing confirm that the signal at certain concrete array position or element place is a true positives or possibly be spurious response.Exemplary chip-reader for example Fodor etc. (U.S. Patent number 5,925,525) is said.Perhaps, when array is when being processed by the microballon mixture of various addressable phenotypic markers, can adopt this reaction of Flow cytometry.

5.2 Protein diagnostic

Consistent with the present invention is, exists the HVI labelled protein of abnormal concentrations to show herpesvirus infection, the particularly existence of reactivity herpesvirus infection, degree or stage, or the risk of herpesvirus infection sequela takes place.Can adopt the imitate level of HVI labelled protein in the article of any appropriate method detection of biological known in the art.For example, when the HVI labelled protein is enzyme, can come this protein of quantitatively determined according to its catalytic activity or contained per sample this protein molecule number.Can adopt antibody technique, for example immunohistology and immunohistochemical method come the level of proteins of interest matter in the test set tissue samples.For example, specific recognition is provided, detects anti-existing (or combination) with second detection system with one anti-(polyclone or mono-clonal).Can be with detectable, but for example fluorescent marker, radioactively labelled substance maybe can produce quantitatively determined (as colored) product enzyme (as, SEAP, horseradish peroxidase) with two anti-couplings mutually.In another suitable method, detectability ground mark one is anti-own.Therefore can make the immunohistology mark to tissue slice.In some embodiments, preparation is analyzed and is used protein extract from biological sample (for example, tissue, cell).Can adopt routine immunization trace method (Jalkanen etc., 1985, J.Cell.Biol., 101:976-985; Jalkanen etc., 1987, J.Cell.Biol., 105:3087-3096) level of proteins of interest matter in western-trace or spot/narrow line experiment (analysis) this extract (for example, washing composition extract).

Other useful antibody method comprises immunoassay, for example enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).For example, the protein specific monoclonal antibody can be used as immunosorbent and enzyme labelled probe detects and the interested HVI labelled protein of quantitatively determined.Can adopt the linear regression computerized algorithm (referring to, Lacobilli etc., 1988, Breast Cancer Research and Treatment, 11:19-30), the content in the reference standard goods comes this Protein content in the calculation sample.Other embodiment can be used two kinds of different monoclonal antibodies of proteins of interest matter, and is a kind of as immunosorbent, another kind of as enzyme labelled probe.

In addition, the latest developments in protein capture array field make and can detect simultaneously and/or a large amount of protein of quantitatively determined.For example, the low density protein array on the filter membrane is like general protein array system (Ge, 2000, Nucleic Acids Res., 28 (2): e3) adopt standard ELISA technology and scanning electric charge coupling device spare (CCD) detector to take the AM picture of forming array.Also developed the immunosensor array that can detect clinical (multiple) analyte simultaneously.Can come in the analysing body fluid with protein array at present, for example the protein expression distribution pattern in the object serum before and after health or ill object and the pharmacological agent.

The protein capture array generally comprises the multiple proteins trapping agent, and they have formed the different element in this array manifold position separately.The protein capture agent can be ability conjugated protein and any molecule or the molecular complex that is fixed in the protein capture agent site on the array.The protein capture agent can be a kind of protein, and its natural function in cell is that specificity combines another kind of protein, for example antibody or acceptor.Perhaps, the protein capture agent can be specificity to combine certain proteinic partial synthesis or protein complete synthesis or reorganization.Perhaps, the protein capture agent can be external basis to the binding affinity of specific target protein matter or peptide from mutagenesis, at random or the protein of selecting completely randomization and the synthetic library.It is methods of exhibiting known in the art that used system of selection can be chosen wantonly, for example ribosomal display or phage display method.Perhaps, the protein capture agent that obtains through external selection can be can specificity combine the DNA of target protein or RNA fit (referring to, Potyrailo etc. for example, 1998, Anal.Chem., 70:3419-3425; Cohen etc., 1998, Proc.Natl.Acad.Sci.USA, 95:14272-14277; Fukuda etc., 1997, Nucleic AcidsSymp.Ser., 37:237-238; Derive from SomaLogic).For example, use Selex ^TMMethod is selected fit from oligonucleotide library, can be through covalently bound, and for example mix bromination deoxyuridine and uv-activated crosslinked (light is fit) and improve they and protein interactions.Fit advantage be to be easy to through automatic oligonucleotide synthesize prepare and DNA stable and firmly; Can adopt general fluorescence protein staining technique to detect the combination situation.Perhaps, the protein capture agent of external selection can be polypeptide (for example, antigen) (referring to, for example Roberts and Szostak, 1997, Proc.Natl.Acad.Sci.USA, 94:12297-12302).

The another kind of array of capture molecules is the array of processing through " molecule printing " technology, peptide wherein (for example proteinic C-stub area) produces complementary structure as template in polymerizable matrix sequence-specific space; These spaces ability specificitys are caught (sex change) protein with corresponding one-level aminoacid sequence and (for example, are derived from ProteinPrint then ^TMWith Aspira Biosystems).

The exemplary proteins capture array comprises that being commonly referred to has living space containing of antibody array and goes up the array of addressable antigen binding molecules, and this array helps on a large scale the numerous protein of parallel analysis to confirm protein group or protein is inferior organizes.Antibody array shows to have required specificity character and acceptable background, and some arrays can be buied (for example, BD Biosciences, Clontech, BioRad and Sigma).The existing report of the whole bag of tricks of preparation antibody array (referring to, Lopez etc. for example, 2003, J.Chromatogr.B, 787:19-27; Cahill, 2000, Trends in Biotechnology, 7:47-51; U.S. Patent Application Publication 2002/0055186; U.S. Patent Application Publication 2003/0003599; PCT announces WO03/062444; PCT announces WO03/077851; PCT announces WO02/59601; PCT announces WO02/39120; PCT announces WO01/79849; PCT announces WO99/39210).The antigen binding molecules of these arrays can be discerned the inferior group of the expressed protein of certain cell or cell mass at least, and its exemplary comprises that growth factor receptors, hormone receptor, neurotransmitter receptor, catecholamine acceptor, amino acid derivative acceptor, cytokine receptor, extracellular matrix acceptor, antibody, lectin, cytokine, silk press down that albumen, proteolytic enzyme, kinases, Phosphoric acid esterase, ras-appearance GTP enzyme, lytic enzyme, steroid hormone receptor, transcription factor, thermal excited transcryption factor, DNA-are conjugated protein, zinc finger protein, leucine zipper protein, homeodomain protein, intracellular signal transduction regulator and effector, apoptosis correlation factor, DNA composition-factor, DNA reparative factor, DNA recombinant factor, cell-surface antigens, hepatitis C virus (HCV) proteolytic enzyme and hiv protease.

In phage display or ribosomal display library (for example; Derive from Cambridge AntibodyTechnology; BioInvent; Affitech and Biosite) in can pass through routine immunization method (for example, polyclonal serum and hybridoma) after selecting, or as the common antigen binding molecules for preparing antibody array at the recombinant fragment of expression in escherichia coli.Perhaps, " combinatorial antibody (combibody) " form that contains non-covalent associating VH and VL structural domain of preparation makes up formed matrix for producing double antibody bacterial clone (for example, deriving from Domantis).Exemplary antigen binding molecules as the protein capture agent comprises monoclonal antibody, polyclonal antibody, Fv, Fab, Fab ' and F (ab ') ₂Immunoglobulin fragment, synthetic stablize Fv fragment (like strand Fv fragment (scFv), through the stable Fv fragment (dsFv) of disulfide linkage), the little antibody of single variable region structural domain (dAbs) (minibody), combinatorial antibody and multivalent antibody (as double antibody with many-scFv), the single structure territory of camel (camelids) or engineered people's equivalence antibody.

It generally is the support surface of putting down or fluctuating is arranged that each different protein capture agent of locus is connected in usually.Physical support thing commonly used comprises slide glass, silicon, micropore, nitrocellulose or pvdf membrane and magnetic micro-beads and other microballon.

Though extensively adopt the protein droplet is delivered on the plane; But also can adopt other relevant structure; The CD centrifugal device that comprises the progress exploitation of instrument Microfluidics (for example; Available from Gyros) and the chip design of specialization, for example engineered microchannel (for example, The Living Chip in the plane ^TM, available from Biotrove) and the small 3D post (for example, deriving from Zyomyx) of silicon face.

Also can the basis of the particle in the suspension-s as array be needed only the identity coding to them; These systems comprise with color-coded microballon (for example, available from Luminex, Bio-Rad and NanomicsBiosystems), semiconductor nanocrystal (for example, Qdots ^TM, available from Quantum Dots), clavate encoded beads (UltraPlex ^TM, available from Smartbeads) and many metals microbot (Nanobarcodes ^TMParticle is available from Surromed).Also can these pearls be assembled into planar array (for example, available from LEAPS technology and BioArray Solutions) on semi-conductor chip.When using particle, each protein capture agent is connected in each particle usually and confirms position or the array that separates so that the space to be provided.Distinguish (but parallel) then with isolation method, for example detect these particles in the hole of titer plate or in the different test tube.

In operation, with the optional fragmentation of protein example with form peptide fragment (referring to, for example U.S. Patent Application Publication 2002/0055186), be suitable for being delivered to the protein capture array under protein or the peptide bonded condition; Wash this array with not combining in the sample or the component of non-specific binding flush away from the array.Then, utilize the protein of each combination of elements on suitable detection system detection and the array or the existence or the content of peptide.To make comparisons with the protein determination amount of certain combination of elements on the array and second Protein content of this array second combination of elements.In certain embodiments, second Protein content is known or known constant in the sample.

For analyzing the difference of protein expression between two kinds of cells or the cell mass, the protein example of first kind of cell or cell mass is being suitable for being delivered to this array under the condition of protein bound.The protein example of second kind of cell or cell mass is delivered to second array identical with first array in a similar manner.Wash two arrays then with not combining in the sample or the component of non-specific binding flush away from the array.At last, make comparisons with keeping keeping with the respective element bonded protein contnt of second array with the first array element bonded protein contnt.For measuring the difference of protein expression mode between two kinds of cells or the cell mass, will with the protein contnt of first each combination of elements of array from the respective element bonded protein contnt of second array deduct.

In an exemplary, fluorescent mark capable of using detects and array bonded protein.Will with understand the used identical instrument application of dna microarray in the protein capture array.For showing difference; The available fluorescent mark protein detection capture array (for example, antibody array) that derives from two kinds of different states cells, the cell lysate of two states is with different fluorophores (for example; Cy-3 and Cy-5) mark and mixing, thus can be with the reading of color as the variation of target (molecule) abundance.Can the sensitivity of fluorescence reading be improved 10-100 doubly through junket acid amides amplification of signal (TSA) (for example, available from PerkinElmer Lifesciences).Slab guide technology (for example, available from Zeptosens) can be carried out overdelicate fluoroscopic examination, and its another advantage is to need not washing.Utilize mark thing (for example, available from Luminex) or utilize the suspended beads and the particle of semiconductor nanocrystal (for example, available from Quantum Dot) performance also can realize highly sensitive of phycoerythrin.Adopted the combination (for example, available from Affibody) that FRET detects the unmarked part that can be used for array.Several kinds of other reading devices have been developed; The modifying device that comprises following method: surperficial plasmon resonance (for example; Available from HTS Biosystems and Intrinsic Bioprobes), rolling circulation DNA cloning (for example, available from Molecular Staging), mass spectrum be (for example, available from Sense Proteomic; Ciphergen; Intrinsic and Bioprobes), resonant light scattering (for example, available from Genicon Sciences) and AFM (for example, available from BioForce Laboratories).NextGen and PerkinElmer Life Sciences have developed jointly and have been used for sample and the automatic microfluid system of cultivating and washing of slide glass array.

In certain embodiments; The technology that is used to detect HVI marker expression product comprises with inside or external perimysium reference article coming quantitatively or those products of semiquantitative determination, thus the level or the functionally active of corresponding expression product in these expression products and the reference sample in the comparative biology sample effectively.The technician adopts standard method can confirm this standard substance.The level of each expression product or the absolute value of functionally active have been measured in the specific embodiment.

In specific embodiment; Capable of usingly await the reply described in the PCT application number PCT/AU03/01517 like what submit in international publication number WO02/090579 and on November 14th, 2003, comprise with the base station mutually the system at least one TS of joining of coupling carry out this diagnostic method.The base station usually with one or more DBs coupling couplet mutually; These DBs contain the representative HVI marker expression product level of a large amount of individualities or the tentation data of functionally active; And when collecting these tentation datas these individual virtual conditions indication (for example, the existence of herpesvirus infection, do not exist, degree, stage or the risk of simplexvirus sequela takes place).In operation; The base station is data through station, network of communication receiving terminal and said data and the tentation data that is stored in the DB made comparisons generally, and said data represented at least a expression product detects or level of standardization or functionally active in the tested object biological sample corresponding to taking from.The data of this object and tentation data made comparisons make the base station can confirm the state of this object according to comparative result.Therefore, the individuality that has the similarity parameter value with tested object is attempted to identify in the base station, in case confirmed state according to this evaluation (result), the TS can be delivered to the diagnosis that shows in the base station.

5.3 Test kit

Can be assembled in the test kit with required all necessary materials and the reagent of quantitatively determined HVI marker gene expression product detecting.These test kits are also optional to be equipped with suitable agent, the positive and negative control, washing soln, blotting membrane, the titer plate dilution buffer liquid etc. of certification mark thing.For example, kit for detecting nucleic acid can be equipped with (i) HVI mark polynucleotide (can be used as positive control), (ii) can with the primer or the probe of certain HVI mark polynucleotide specific hybrid.Also the enzyme that is applicable to amplification of nucleic acid can be housed, comprise various polysaccharases (reversed transcriptive enzyme, Taq, Sequenase ^TMDna ligases etc. depend on the nucleic acid amplification technologies that is adopted), deoxynucleotide and damping fluid are to provide amplification required reaction mixture.This test kit generally is equipped with the different vessels of all ingredients and enzyme and various primer or probe with suitable method.Perhaps, protein detection reagent kit can be equipped with: immune interactional antigen binding molecules, (ii) can take place with certain HVI mark polynucleotide in (i) HVI labeling polypeptide (can be used as positive control).The characteristic of this test kit also is to be equipped with various devices and the reagent that carries out test described herein; And/or the working instructions of the printing of expressing with this test kit quantitatively determined HVI marker gene.

These test kits are optional to be equipped with suitable agent, the positive and negative control, washing soln, blotting membrane, the titer plate dilution buffer liquid etc. of certification mark thing.For example, kit for detecting nucleic acid can be equipped with (i) HVI mark polynucleotide (can be used as positive control), (ii) can with the primer or the probe of certain HVI mark polynucleotide specific hybrid.Also the enzyme that is applicable to amplification of nucleic acid can be housed, comprise various polysaccharases (reversed transcriptive enzyme, Taq, Sequenase ^TMDna ligases etc. depend on the nucleic acid amplification technologies that is adopted), deoxynucleotide and damping fluid are to provide amplification required reaction mixture.This test kit generally is equipped with the different vessels of all ingredients and enzyme and various primer or probe with suitable method.Perhaps, protein detection reagent kit can be equipped with: immune interactional antigen binding molecules, (ii) can take place with certain HVI mark polynucleotide in (i) HVI labeling polypeptide (can be used as positive control).The characteristic of this test kit also is to be equipped with various devices and the reagent that carries out test described herein; And/or the working instructions of the printing of expressing with this test kit quantitatively determined HVI mark polynucleotide.

6. treat or prevention method

Make object have herpesvirus infection sequela risk positive diagnosis takes place after, the present invention is also extensible to be used to treat or the herpesvirus infection, particularly reactivity herpesvirus infection of object of prevention.Generally, said treatment comprises the medicine of diagnosing male object significant quantity or treatment improving symptom or to reverse the generation of herpesvirus infection, or reduces the possibility of object generation herpesvirus infection sequela.The medicine that is applicable to the treatment herpesvirus infection at present includes but not limited to: for example U.S. Patent Application Publication No. 20020147210 described acyclovirs, Famciclovir, valacyclovir, ganciclovir, penciclovir, Zidovodine, cytidine arabinose glycosides, ribavirin, amantadine, iododeoxyuridine, poscarnet, trifluoridine, Metisazone (methizazone), vidarabine, levanisole4-amino-α; Alpha-alpha-dimethyl-2-ethoxyl methyl-1H-imidazoles (imidazo) [4,5-c] quinoline-1-ethanol; U.S. Patent Application Publication No. 20020103262 described hydroxylation tolane; U.S. Patent number 5,840,728 described cyclopropylization (cyclopropanated) carbocyclic rings 2 '-deoxynucleoside; U.S. Patent number 6,048,843 described thymus pyrimidines-analogue herpes medicine; (PFA originates from Astra to U.S. Patent Application Publication No. 20040053891 described phosphine formic acid, Sodertlje, the Foscavir of Sweden ^TM), 5-(E)-bromo vinyl uracil analogues and relevant pyrimidine nucleoside; U.S. Patent Application Publication No. 20040024209 described 1-aryl-4-oxygen-1,4-dihydro-3-quinolinecarboxamideas; With Raymond etc., (1984, Biochem.Bioph.Res.Comm., 121 (3): 848-854) described spermidine catechol acid amides iron chelating agent.

Perhaps, capable of using like U.S. Patent Application Publication No. 20020099426 described device treatment targets, said device sends electricity irritation to infected skin of patient or mucous membrane.A series of electricimpulses to have the different electric characteristic apply electricity irritation.

However, it should be understood that the present invention includes to can be used for treating or any medicine or the method for prophylaxis of herpes viral infections, be not limited to above-mentioned exemplary compound and preparation.

The simplexvirus cushion generally becomes medicine (or veterinary science) compsn with pharmaceutically acceptable vehicle group, realizes the purpose that they are required with significant quantity.The dosage that gives the active compound of object should be enough in object, cause useful reaction in for some time, for example reduced or alleviate herpesvirus infection, particularly the symptom of reactivity herpesvirus infection.The consumption of the pharmaceutically active compound that gives depends on object to be treated, and comprises its age, sex, body weight and whole body health situation.The accurate consumption of the active compound that is given thus, depends on the doctor's that obtains employment judgement.Confirming to treat or during the significant quantity of the active compound of prophylaxis of herpes viral infections, doctor or animal doctor should assess with herpesvirus infection and have related indication seriousness, comprise above-mentioned herpesvirus infection sequela relevant symptoms.In any situation, those skilled in the art are not difficult to confirm appropriate dose and the suitable regimen of herpesvirus infection cushion and need not too much experiment.

In specific embodiment, there is EHV or after its stage at the positive object of diagnosis, the present invention is extensible to be used to treat that EHV infects, treatment EHV infection and recurrence or object of prevention are further infected by EHV.Generally, treatment comprises to be isolated in case further infect, and the palliative treatment damages avoiding with having a rest over a long time.The present invention can make more effective isolation and control decision when animal is infectivity; Thereby the animal doctor, horse owner people and the trainer that know this information just can stop protecting from infection other horses, particularly other pregnant mare until the virus diffusion with these animals isolation.On the contrary, existing method can only could be diagnosed EHV later at infection stage warp, therefore can not be used for isolating decision.

For ease of understanding and embodiment of the present invention, particularly preferred embodiment has been described through following non-limiting example.

Embodiment

Embodiment 1

Identify the specific diagnosis gene of herpesvirus infection

Experimental disease test design

At different time, in two groups of 13 young coltfoals, induce equid herpesvirus 1 (EHV-1) to infect.6 young coltfoal tracing observations 20 days (group 1) infected at the 0th day (experimental inoculation).7 young coltfoal tracing observations 42 days (group 2) infected at the 21st day (experimental inoculation).All young coltfoals of EHV-1 nasopharynx aerosol infection with vitro culture.Group 1 blood sample was 8 time point collections of the 0th, 1,2,4,6,10,13 and 20 day.Group 2 blood sample was 16 time point collections of the 0th, 1,2,4,6,10,13,20,21,22,23,25,27,31,34 and 41 day.The 0th day sample is as the contrast of each dry goods.

The animal of discovery group then 2 has received natural and nonsynchronous infection of EHV.All animal serums sun of group 2 changes, and utilizes the time (before seropositive conversion 10-14 days) of seropositive conversion time estimation natural infection.Because group 2 infection time is asynchronous, the diagnostic characteristic that utilizes the gene expression datas of these animals to check utilization group 1 to obtain.

Carry out following check and observation at all time points:

● health check-up comprises body temperature, pulse and respiration measurement

● hematology and biological chemistry (check)

● nose swab is carried out PCR

● serum EHV-1 antibody (Ab) titre

● leukocyte specific gene array is carried out gene expression analysis.

Utilization contains the GeneChips of thousands of kinds of expressed genes of horse white corpuscle ^TM(method of use is specified in hereinafter " generation gene expression data ") analyzed the blood sample of taking from infected animals.Analyze these data (" identifying response gene and proof diagnosis potentiality " vide infra) disclose infect before and after the EHV and back first day of infection between many genetic expressions of animal variant.Can design in a kind of test test sample at least a; The rna level of preferred at least two kinds of HVI marker gene, the representative transcription sequence of said gene is as follows: 1,2,4,6,7,8,10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113.The specificity and the level of sensitivity of this method are 94%.Perhaps, any combination of one of at least two kinds of polynucleotide and listed other the 61 kinds of HVI mark polynucleotide of table 1 all has strong diagnosis capability.

Materials and methods

Blood sampling

Extract high-quality RNA or protein from horses (non-affective state) blood sampling.The suitable blood sampling test tube of collection, preservation, transhipment and isolation of RNA comprises PAXgene ^TMTest tube (PreAnalytix Inc., Valencia, CA, the U.S.).Perhaps, can blood collecting be gone into to contain to be designed in the test tube of solution (available from Roche, Ambion, Invitrogen and ABI) of preservation nucleic acid.Be to measure protein level, the 50ml blood collecting is gone into to contain in the test tube of 4ml4% Trisodium Citrate in case condense.Separate white corpuscle and blood plasma, freezing is used for subsequent analysis and detection specificity protein.The PAXgene test tube is remained in room temperature, extract RNA then.With standard format record clinical sign.

The test kit of Qiagen Inc (Valencia, CA, the U.S.) be equipped with the 2.5mL blood of collection PAXgene blood rna test tube (PAXgene Blood RNA Tube) with reagent that separates total RNA and working instructions.Separation begins to precipitate the nucleic acid the PAXgene blood rna test tube from centrifugation step.Washing precipitate, resuspended, cultivate with digesting protein with the optimization damping fluid that contains Proteinase K.Recentrifuge is removed residual cell debris, and supernatant is transferred in the fresh micro-centrifuge tube.Add ethanol and regulate the combination condition, lysate is put on PAXgene RNA spin (spin) post.During simple centrifugal, RNA optionally combines with pellosil, and pollutent flows out.Remove residual pollutent through three effective washing steps, use buffer B R5 eluted rna then.

Need before the test with Agilent biological analyser and spectrophotometer with absorbancy 260/280 ratio quantitative and qualitative test RNA.

DNA extraction

(Valencia, CA, test kit USA) are equipped with the PAXgene blood DNA test tube (PAXgene Blood DNA Tube) and reagent that separates total DNA and working instructions of the 8.5ml blood of collection to Qiagen Inc.It is centrifugal then that separation begins to add extra cracked solution.Washing precipitate, resuspended is cultivated with digesting protein with the optimization damping fluid that contains Proteinase K.Ethanol sedimentation DNA, centrifugal again primary sedimentation nucleic acid.Residual contaminants is removed in washing, then DNA is resuspended among the buffer B G4.

Need be before the test with spectrophotometer or agarose gel electrophoresis quantitative and qualitative test DNA.

Serum antibody is measured

Basically like Foote etc., (Equine Vet J., 36 (4): 341-345,2004) are said, measure the serum antibody level of EHV-1 through ELISA with reorganization EHV-1 gp G.Total RNA extracts.

Produce gene expression data

Method is selected

Can adopt the specific RNA level in the various technology for detection tissue samples.Two kinds of routines well known in the art and wieldy technology are:

Adopt the Affymetrix technology to make GeneChip ^TMAnalyze;

Real-time polymerase chain reaction (for example, the TaqMan of Applied Biosystems ^TM).

GeneChips ^TMCome quantitatively determined RNA through detecting with the mark cRNA that is structured in the short oligonucleotide hybridization on the silicon substrate.This technology and method see www.affymetrix.com for details.

Real-time polymerase chain reaction (RT-PCR) utilizes the probe of two kinds of PCR primers, mark and heat-stable DNA polymerase to come quantitatively determined RNA.The color that the PCR product produces is released into solution, detects this color.Internal contrast commonly used, the for example original level of total RNA in the 18S rna probe working sample.Measure each gene and internal contrast respectively.This technology and method see www.appliedbiosytems.com or www.qiagen.com or www.biorad..com for details.AppliedBiosystems provides following service: promptly the client provides dna sequence dna information and negative expense, provides as feedback the said firm each gene is carried out the required whole reagent of RT-PCR.

GeneChip ^TMThe advantage of analyzing is once can analyze thousands of genes.Yet this analysis is expensive and carry out a test and take more than 3 days.RT-PCR generally once can only analyze a kind of gene, but cheap and can in one day, accomplish.

If concrete number gene to be analyzed is less than 20, then RT-PCR is optional gene expression analysis method.When needs are analyzed many genes simultaneously, GeneChip ^TMOr other gene expression analysis technology (for example Illumina pearl array) is optional method.

Be used for producing and analyzing GeneChip ^TMThe method and the PCR in real time of data are summarized in hereinafter.

Produce GeneChip ^TMData

Preparation cDNA and cRNA

Prepare the method that the following method of cDNA and cRNA adopts Affymetrix (www.affymetrix.com) to provide and recommend from total RNA.

Step is:

Total RNA of 3 μ g is as the double-stranded cDNA of template for preparing altogether.

Preparation cRNA also marks with biotinylation uridylic (dUTP).

The cRNA that purifying (clean) vitamin H is marked utilizes spectrophotometer and the quantitatively determined of MOPS gel analysis.

The cRNA fragment of marking is turned to about 300bp size.

Utilize Agilent " Lab-on-a-Chip " system (Agilent Technologies) to measure rna content.

Hybridization, washing and dyeing

Step is:

Preparation contains 0.05 μ g/ μ L marks and the cRNA of fragmentation, the hybridization mixture that doping (spike-in) positive is hybridized contrast and Affymetrix oligonucleotide B2, bioB, bioC, bioD and cre.

The hybridization mixture of final volume (80 μ L) is added GeneChip ^TMIn the medicine box.

This medicine box is placed the hybrid heater of constant rotational speed, 16 hours.

Remove GeneChip ^TMIn liquid and preserve.

With GeneChip ^TMPlace the fluid station.

With each GeneChip ^TMExperiment condition be recorded as the .EXP file.

After the helpmate added suitable solution, all washing and dyeing course were carried out in Affymetrix fluid station.

Washing GeneChip ^TM,, wash once more with low salts solution then with Streptavidin-phycoerythrin dyeing.

Washing " excites " dyestuff on the probe array with laser after accomplishing, and passes through CCD camera image with Affymetrix scanner (Agilent manufacturing).

Scanning and generation data file

Scanner and MAS5 software produce a GeneChip ^TMImage file, be called .DAT file (referring to the figure at the back side).

Carry out statistical analysis behind the pre-treatment .DAT file.

The pre-treatment step of data (before any statistical analysis) comprising:

● the quality control of .DAT file (QC).

Produce the .CEL file.

Demarcate (scaling) and stdn.

.DAT the quality control of file

.DAT file is a kind of image.The human factor (artifact) of manual these images of inspection (for example, height/low strength spot, cut, high local background or total background).(through changing intensity mode that produces the border and the cross performance that the array title is not difficult to identify the B2 oligonucleotide arrays).Thereby MAS5 software utilizes B2 oligonucleotide border to compare the oligonucleotide that each grid could concentrated and identify to grid on the image.

Utilize other doping (spiked) hybridization contrast (bioB, bioC, bioD and cre) through reading the hybridization efficiency of assessing sample with " basis " gene test introducing sub-routine of their relative concentrations of increase reflection of signal value.(if the .DAT file has suitable quality, is translated into intensity data file (.CEL file) with Affymetrix MAS5 software).

Produce the .CEL file

The .CEL file that produces from the .DAT file with MAS5 software comprises the green strength that this probe groups is calculated.The background value that deduction calculates from each cell (mensuration) value obtains gene expression data.For removing negative intensity level, used noise compensation fraction based on the local noise value of the standard deviation of minimum 2% background value.

With GeneChip ^TMAll .CEL file applications that produce are in specific quality metric canonical parameter.

Some modules are recommended by Affymetrix is conventional, can be used as GeneChip ^TMA part and the Affymetrix internal contrast that provides is measured.Other module rule of thumb with many GeneChip ^TMProcessing and decide.

Analyze GeneChip ^TMData

Three kinds of exemplary method that can be used for data normalization are:

Affymetrix MAS5 algorithm.

The reliable multicore sheet of Irizarry is analyzed (Robust Multi-chip Analysis) (RMA) algorithm (Irizarray etc., 2002, Biostatistics (in the printing)).

● reliable multicore sheet is analyzed reserving model (Robust Multi-chip Analysis Saved model) (RMAS)

Those skilled in the art should know and can adopt many other methods and can materially affect not arranged to the present invention.

Affymetrix MAS5 algorithm

Affymetrix MAS5 software utilizes the .CEL file to come stdn or nominal data.The nominal data of chip piece is made comparisons with the similar nominal data of another chip.

Acquiescence " whole world calibration (Global Scaling) " option to .CEL file applications MAS5 algorithm is realized Affymetrix MAS5 stdn.This method has been deducted the reliable estimated value of probe value distribution center, again divided by the variable reliable estimated value of probe.This has produced a core assembly sheet that has common location and demarcation in the probe level.

All probes through to certain given gene obtain gene expression index to reliable equalization method.The result is forced to non-feminine gender.

In view of being to demarcate, so even overall gene expression dose also possibly there are differences between chip after the stdn in the probe level rather than at gene level.After standard MAS5 stdn, make each genic value go trend (de-trend) according to chip intensity intermediate value.That is, each genic value returns according to chip intensity intermediate value, calculates residual error.Get these residual errors and go the estimated value of trend as each genetic expression.

With Affymetrix MAS5 arithmetic calculation chip median intensity, but calibration factor fixedly is one.

RMA analyzes

Except setting up probe weight and target fractile (quantile) with the long-term library of chip.cel file and not to these specificity chips again outside the double counting, this method is identical with the RMA method.The probe level is carried out stdn once more.

Produce the PCR in real time data

(2000, J Mol Endocrinol can obtain the background information of executive real-time PCR in 25:169-193) in the summary of for example http://dorakmt.tripod.com/genetics/realtime.html and Bustin SA.

TaqMan ^TMPrimer and probe design guide:

1.Primer Express ^TM(ABI) software design melting temperature(Tm) (Tm) be the probe more than 10 ℃ for 58-60 ℃ primer and Tm value.The Tm of two kinds of primers should be identical;

2. primer should a long 15-30 base;

3.G+C the preferred 30-80% of content.Can't avoid if G+C content is higher, then need adopt high annealing and melting temperature(Tm), use cosolvent, for example glycerine, DMSO or 7-denitrogenation are mixed-dGTP;

4. should avoid the identical Nucleotide of bunchiness.This is even more important as far as G, and the G string more than 4 cannot be arranged;

5. the sum of G and C should be no more than 2 (this software of more recent version has the option that automatically performs this function) in last 5 Nucleotide of primer 3 ' end.Thereby this helps to reduce non-specific sensitization in 3 ' the terminal relative instability of introducing of primer.The primer condition is identical with SYBR Green test;

6. the length of maximum amplicon should not surpass 400bp (preferred 50-150 base).Less amplicon can obtain more consistent result, requires the short function with 5 ' nuclease of length irrelevant because PCR more tolerates more effectively and to reaction conditions);

7. this probe should not contain the identical Nucleotide (the particularly continuous G more than 4) of bunchiness, and G+C content should be 30-80%, and C should be more than G, and 5 ' end should not be G.The quantity of C is many more, and the Δ Rn of generation is high more.Should select probe earlier;

8. be the false positive due to the contaminative genomic dna in the cDNA preparation of having avoided gene-amplification, primer should be crossed over exon-exon joining region.So can amplifying genom DNA (the PDAR test kit that is used for people GAPDH amplification is equipped with this primer);

9. if design TaqMan ^TMProbe is distinguished allelotrope, and the Nucleotide of mispairing (pleomorphism site) should be at probe centre rather than two ends;

10. near the primer that contains dA Nucleotide the 3 ' end capable of using passes through AmpErase ^TMDegrade effectively (see EZ RT-PCR test kit handbook the 9th page of any primer dimer of being produced of UNG; P/N402877).If can not be chosen near the primer that contains dA Nucleotide two ends, then the considered utilization has the primer of 3 ' terminal dU-Nucleotide.

(also can referring to the general principle of " PCR design of primers " (PCR Primer Design) of Invitrogen).

Universal method:

1. (not containing oligomerization-dT) is cDNA with total RNA rt with random hexamer.If have to oligomerization-dT, then should avoid the upper reaches that two kilobase above long mRNA transcript or amplicon are arranged, 18S RNA can not be used as standard substance;

2. multiplex PCR only has in limited time at the contrast primer and can correctly carry out (primer that the ABI contrast agents does not limit them);

3. target cDNA amount ranges is 10ng-1g.If with DNA (being mainly used in the allelotrope Study on Identification), optimum amount is 100ng-1g;

4. the most handy DNA enzyme that does not contain the RNA enzyme is handled each RNA goods and is polluted to avoid genomic dna.Even best RNA process for extracting also can produce some genomic dnas.Certainly the not primer of amplifying genom DNA is preferably arranged, but this does not accomplish sometimes;

5. for obtaining optimum, reagent (before preparation PCR mixture) and PCR mixture itself (before the application of sample) should fully vibrate (vortex) with mix.Otherwise the Rn value possibly have drift in the early stage round (0-5) of PCR.Earlier with probe add the damping fluid component make its at room temperature balance the reagent preparation mixture is also very important again.

TaqMan ^TMPrimer and probe:

Order TaqMan from ABI medium-scale (midi-scale) ^TMIt when probe is received the suspension of 100 μ M.If prepare 1/20 diluent, then obtain 5 μ M solution.This stock solution should be divided into equal portions, freezing, black out preservation.Its consumption is the 100nM final concentration that 1 μ L obtains recommending in 50 μ L reaction volumes.

Be freeze dried when primer is received, content is marked on (for example 150.000pmol equals 150nmol) on the test tube with pmol.If the primer of X nmol is resuspended in the X μ L water, the solution that obtains is 1mM.It is freezing preferably this storing solution to be divided into equal portions.When the 1mM storing solution was diluted 100 times, the working solution that obtains was 10 μ M.For making the 50-900nM of final primer concentration for recommending in the 50 μ L reaction volumes, 0.25-4.50 μ μ L (the 500nM final concentration is with 2.5 μ μ L) is used in each reaction.

PDAR primer and probe provide to be stored in a mixture in the test tube.Their consumptions in 50 μ L reaction volumes are 2.5 μ L.

One step TaqMan is set ^TMReaction:

One step PCR in real time is used as template with RNA (opposite with cDNA).Preferred this method if the RNA strength of solution is low, but only when carrying out the substance reaction.Shortcoming is that the RNA hangover makes enzyme AmpErase can not be used for single step reaction.In this method, rt and PCR in real time occur in the same test tube.The downstream PCR primer also is used as the primer (random hexamer or oligomerization-dT can not be used for the rt of an one step RT-PCR) of reversed transcriptive enzyme.Single step reaction needs higher dNTP concentration (more than or equal to 300mM and 200mM), because this reaction has merged a kind of of two kinds of reactions needing dNTP.It is following that one of Gold RT-PCR test kit goes on foot the used type reaction mixture of PCR:

If use PDAR, then use primer+probe mixture of 2.5 μ L.

10pg-100ng RNA is preferably used in this reaction.Attention is reduced to 50ng with the template consumption from 100ng will make the CT value increase by 1.For making the CT value reduce by 3, initial template consumption should increase by 8 times.ABI declares that this system can detect the RNA of 2 piks, and the maximum consumption of RNA can be 1 microgram.For carrying out routine analysis, the RNA of available 10pg-100ng and 100pg-1 μ g genomic dna.

The loop parameter of one step PCR:

Rt (MuLV), 48 ℃ 30 minutes.

The AmpliTaq activation, 95 ℃ 10 minutes.

PCR:95 ℃ of sex change 15 seconds, 1 minute (repeating 40 times) (on ABI7700, the minimal maintenance time is 15 seconds) of 60 ℃ of annealing/extensions.

The EZ one-step of up-to-date introduction ^TMRT-PCR test kit UNG capable of using is because utilized the thermostability reversed transcriptive enzyme to make that the cultivation time of rt is 60 ℃.This temperature also is the better selection of avoiding in 48 ℃ of formation primer dimers and non-specific binding.

Operation A BI7700:

Should confirm following item before bringing into operation:

1. the loop parameter of operation is correct;

2. the selection of spectrally compensating correct (off is selected in the substance reaction, and multiple reaction selects on);

3.Analysis " Number of PCR Stages (the PCR stage numbers) " among the Options box (analysis option frame) (Analysis (analysis)/Options (option)) selects correct.If do not have data among the amplification figure, but visible in plat, and the X-axle indication range 0-1 of amplification (figure) wheel, then after operation once, must manually set this (parameter).

4. template contrasts (Template Control) mark (being accurate calculation Δ Rn) like this;

5. should correctly select dye component before the data analysis;

6. you must entitle (can not keep unnamed) before the operation beginning and preserve;

7. when end of run, preserve data earlier and begin again to analyze;

It is extremely careful that (use) ABI software needs.Press down do not attempt behind Run (operation) button out of service.You have problem and if desired during the switch machine, must grade just can restart operation at least 1 hour.

When analytical data, remember that the baseline setting of giving tacit consent to is 3-15.If any CT value 15, should change baseline (baseline stop to be worth should than the little 1-2 of CT value of minimum) thereupon." ABI baseline and threshold value are provided with guide " (ABI Tutorial on Setting Baselines andThresholds) seen in the useful discussion of this problem.(interesting is that TaqMan is seen in the best discussion of this problem ^TMIn the handbook of human endogenous control board (Human Endogenous Control Plate)).

If the result is nonsensical, the inspection original spectrum sees that run duration CDC photographic camera (whether) maybe be saturated.Utilize optical cover (optical cap) rather than optics viscosity cover (optical adhesive cover) can prevent that the CDC photographic camera is saturated.When utilizing SYBR Green I, multiple (reaction) and higher concentration probes, (the CDC photographic camera is saturated) more possibly take place.

The result resolves:

When each reaction finished, the fluorescence intensity that is write down was used for following calculating:

Rn ⁺It is the Rn value of reacting when containing all components; Rn ^-It is the Rn value (value that detects among baseline value or the NTC) of unreacted sample.Δ Rn is Rn ⁺With Rn ^-Between difference.It is the number of signals magnitude index that PCR produces.

Quantitatively determined template content has three kinds of exemplary method:

1. absolute standard method: in this method, with the known standard substance of content, for example in vitro translated RNA (cRNA).

2. relative standard's method: the target nucleic acid that comprises known quantity in the test design of each run;

3. comparative CT method: this method is without the known standard substance of content, but the relative quantity of target sequence and any reference value of selection are made comparisons, the amount that the result provides with respect to reference value (the for example expression level of tranquillization lymphocyte or standard cell lines system).

The comparative CT method (Δ Δ CT) that the genetic expression relative quantification is measured:

This method need not production standard curve quantitatively determined template relatively and improves quantity of sample handling when the expression level of observing with respect to active reference control (standard substance).Be successful implementation this method, should make the dynamicrange of target and reference substance similar.The sensitive method of controlling this (dynamicrange) is to observe Δ C _THow (same starting template consumption is made the difference between two CT values of twice PCR gained) change with the extent of dilution of template.If the efficient of two kinds of amplicons about equally, with the logarithm and the Δ C of input _TMapping obtains the line (slope near level<0.10).It is identical that this is illustrated in the twice PCR efficient of carrying out in the starting template amount ranges.If it is unequal to scheme display efficiency, should adopt the typical curve method to come quantitative measurement of gene expression.The accurate minimum and maximum concentration that should measure (1) target result and (2) two kinds of gene content results accurate minimum and the dynamicrange of the two the most at high proportion.In conventional competitive RT-PCR, this dynamicrange is limited in target-competition thing ratio and is about 10:1-1:10 (ratio of 1:1 can obtain best particularity).PCR in real time can be realized dynamicrange widely.

Amplification target and endogenous control in different test tubes adopt the typical curve method need to optimize and verify hardly.Adopt the advantage of comparative CT method to be need not production standard curve (sample more hole capable of using).The negative impact of issuable any dilution error when this method has also been eliminated production standard curve sample.

As long as target has similar dynamicrange with standard substance, comparative CT method (Δ Δ CT method) is the most practical method.Estimate the expression level high (therefore, CT value lower) of standard substance than target.Begin quantitative Analysis from the difference (Δ CT) between the CT value that obtains target and standard substance:

Δ CT=CT (target)-CT (standard substance)

This value of quantitative each sample is treated in calculating, and (only if the expression level of target is higher than standard substance, this value should positive value.If feminine gender is also harmless).Relatively should select one of these samples as reference article (baseline) at every turn.Comparative Δ Δ CT calculates and comprises the difference that finds between each sample Δ CT and the baseline Δ CT.If baseline value is represented minimum expression level, estimate that then Δ Δ CT value is negative (because the Δ CT of baseline sample has maximum CT value but maximum because of it).Increase if express in some samples, and reduce in other, Δ Δ CT value will be the mixed number of negative and positive numerical value.The final step of quantitatively determined is that these numerical value are converted into absolute value.This calculation formula is:

Comparative expression level=2-Δ Δ CT

With regard to expression, compare with baseline values, (sample) increases about 23=8 doubly, and reduces the 2-3=1/8 that is about reference article level.Through import simply the CT value with the Excel of Microsoft carry out these calculating (ABI at http://www.appliedbiosystems.com/support/tutorials/7700amp/ relevant for utilizing extension layer program (spread sheet program) to produce the online guide of amplification figure; TaqMan ^TMHuman endogenous control board (Human Endogenous Control Plate) method also comprises the relevant detailed description of carrying out the PCR in real time data analysis with MSExcel).

Is other (definitely) quantivative approach summarized in ABI user's bulletin (http://docs.appliedbiosystems.com/search.taf? _ UserReference=A8658327189850A13A0C598E).Bulletin #2 and #5 are to complete understanding PCR in real time and quantitatively the most useful.

Recommend method:

1. utilize normotopia displacement liquid pipe (positive-displacement pipette) to avoid moving the liquid out of true;

2. the sensitivity of PCR in real time can detect the target among the total RNA of 2pg.The copy number that reacts used total RNA preferably should promptly be enough to produce signal (preferably being lower than 100ng) through the 25-30 wheel.For realizing that this purpose should reduce or increase consumption;

3. the optimum concn of each reagent is following;

I. the concentration of magnesium chloride should be 4-7mM.As far as being 5.5mM with Primer Express software (primer expression software) designed primer/probe the best;

Ii. except that dUTP (if you are using), the concentration of each dNTP is answered balance.Replace the dTTP twice that to control the residual dUTP of the needs concentration of PCR product be other dNTP with dUTP.Though the optimum range of dNTP concentration is 500 μ M-1mM (one step RT-PCRs), the various dNTP of typical TaqMan reaction (just PCR) adopt 200 μ M (dUTP of 400 μ M);

Iii. in per 50 μ L reactants, add 0.25 μ L (1.25U) AmpliTaq archaeal dna polymerase (5.0U/ μ L) usually.This is minimum requirements.If desired, can this amount be increased 0.25U and reach optimum;

Iv. best concentration and probe concentration is 50-200nM, and primer concentration is 100-900nM, tackle three kinds of differing tempss (TaqMan primer 58,60 and 62 ℃) and three kinds of concentration (50,300,900nM) various Combinatorial Optimizations each to primer.This means to be provided with different three groups (three kinds of temperature) that every group is utilized the target template of fixed amount that nine kinds of reactions (50/50mM, 50/300mM, 50/900,300/50,300/300,300/900,900/50,900/300,900/900mM) are arranged.If desired, can (carry out) second takes turns to optimize and improves the result.Selection can provide the primer concentration of minimum CT and the highest Δ Rn to realize optimum performance.Similarly, should concentration and probe concentration be optimized for 25-225nM;

4. if with AmpliTaq Gold archaeal dna polymerase, need be for realizing this purpose in 92-95 ℃ of heating 9-12 minute before PCR.If with AmpliTaq Gold archaeal dna polymerase, need be in reaction on ice.Typical TaqMan reaction comprises that 50 ℃ of UNG (vide infra) cultivated 2 minutes; 95 ℃ of activated polymerization enzymes 10 minutes; Take turns and 60 ℃ 1 minute (annealing and extension) with 95 ℃ of (sex change) 40 in 15 seconds.If parent material is total RNA, before TaqMan reaction, should carry out the rt circulation usually, this circulation (cDNA is synthetic) is by 25 ℃ 10 minutes (primer cultivations), 48 ℃ 30 minutes (carrying out rt with conventional reversed transcriptive enzyme) and 95 ℃ 5 minutes (deactivation reversed transcriptive enzyme);

5. in this reaction, add AmpErase uridylic-N-glycosylase (UNG) and remove any uridylic of mixing in the amplicon in case residual PCR product increases again.This is a reason of using dUTP rather than dTTP in the PCR reaction why.UNG does not cut the single stranded DNA that contains terminal dU Nucleotide inoperative more than 55 ℃.Only if rt and PCR adopt the rTth archaeal dna polymerase, an one step RT-PCR should not use the main mixture (TaqMan EZ RT-PCR test kit) that contains UNG;

6. must comprise in every deblocking reaction flat board at least three parts of non-amplifications contrasts (NAC) and three parts of non-template contrasts (NTC) (for the target amplification of confirming+/-reach 99.7% confidence level in the threshold value, must carry out sextuplicate NTC).The NAC model contains sample, does not contain enzyme.Need to get rid of in the sample or the existing of fluorescence pollutent (these pollutents can cause false positive) in the heat block (heating block) of thermal cycler.If possibly there is the fluorescence pollutent greater than NTC in the absolute fluorescence of NAC in the sample or in the heat block of thermal cycler behind the PCR.

7. if Δ Δ CT method is used for relative quantification measures, should verify the dynamicrange of primer/probe system and standard substance thereof.Realize this verification through carry out (in triplicate) reaction with 5 kinds of RNA concentration (for example, 0.80pg/ μ L, 400pg/ μ L, 2ng/ μ L and 50ng/ μ L).For identical total rna concentration scope, logarithm and the CT value mapping (typical curve) of carrying out the initial content that the real-time RT-PCR of target and standard substance obtains should be (approaching) straight lines;

8. passive reference substance is the dyestuff (ROX) (being present in the TaqMan universal PC R master mixture) that adds in the reaction.It does not participate in the reaction of 5 ' nucleicacidase.It provides the inside reference substance of background emission fluorescence.Available its comes stdn acceptor-dye signal.To (concentration or volume difference) between the Kong Yukong or the non-PCR fluorescence associated fluctuation that change to take place in time carry out this stdn, be different from the stdn that cDNA consumption or PCR efficient are carried out.The emitted luminescence intensity of report dyestuff is carried out stdn divided by the emitted luminescence intensity of passive reference substance.The ratio that obtains is defined as Rn;

9. if carry out multiple (reaction), abundanter target will exhaust all reacted constituents before other target is able to increase.For avoiding this kind situation, should limit the concentration of its primer to abundanter target material;

10.TaqMan universal PC R master mixture should be stored in 2-8 ℃ (not being-20 ℃);

11. report the GAPDH probe that dye marker TaqMan Gold RT-PCR test kit provides with JOE, with VIC mark Pre-Developed TaqMan ^TMThe same probe that Assay Reagents (PDAR) test kit provides.The primer of these people GAPDH test of design can amplifying genom DNA;

12. residual for preventing enzyme, need an one step RT-PCR method of 48 ℃ of cultivations can not utilize AmpErase UNG, but available in the EZ RT-PCR test kit;

13. substance reaction can only be used an one step RT-PCR method, it (is not random hexamer or oligomerization-dT) that the rt method can only be selected downstream primer;

Control and move the liquid error 14. preferably carry out duplicate (reaction), but this unavoidably can increase cost;

15. if, before operation, should verify spectrally compensating option (in AdvancedOptions (Advanced Options)) carry out multiple (reaction);

16. utilizing the passive reference substance (ROX) in the reaction is diverse ways with fluorescence fluctuation (value) stdn with endogenous control (for example GAPDH, active reference substance) with the efficiency standardization of cDNA/PCR consumption;

Also can be used for terminal point PCR 17.ABI7700 not only can be used for quantitative RT-PCR.The latter comprises existence/do not exist test or allelotrope telling test (for example SNP somatotype);

18.PCR former wheel the (0-5 wheel) in the Rn value drift to represent to react all components uneven at first, but do not influence net result, as long as reset the lower value of baseline scope;

19. if notice amplification figure unusual (CT value < 15, in former the wheel, detect the round of amplified signal), should reduce the higher limit of baseline scope, answer dilute sample to improve CT value (pollution also possibly improve the CT value);

20. Δ Rn value low the CT value of expectation (or be higher than) shows that PCR efficient copy number not good or target is low;

21.CT the standard deviation of value>0.16 show and move the liquid out of true;

22. the color green clauses and subclauses of SYBR (SYBR Greenentry) that are provided with in (Pure Dye Setup) of pure color are abbreviated as " SYBR " with capitalization.Any other abbreviation or lowercase can have problems;

23.ABI7700 SDS software have with 8.1 editions Macintosh os and conflict.Be not taken in analytical data on this computingmachine.

24.ABI7700 do not answer Long-time Shut-down.If it is closed, before operation, answer preheating at least 1 hour.Recommendation is being driven instrument at any time, and this is useful to laser.If just start shooting before the operation, the frame of makeing mistakes of display operation system version conflict may appear.If this situation takes place, select " AutoDownload (downloading automatically) " option.

25.ABI7700 a kind of in the real-time PCR system that just can buy, other comprises the system of BioRad, Cepheid, Corbett Research, Roche and Stratagene.

Gene type assay

Many methods can be used for the genotype of analyzing DNA.The summary of allelotrope discrimination method is seen (Biotechniques, 30 (2): 318-322, (2001)) such as Kristensen.This paper has only described a kind of method, allele-specific PCR method.

Design of primers

The computer program that arbitrarily obtains capable of using, for example Primer3 (http://frodo.wi.mit.edu/primer3/primer3_code.html) designs concrete allelic specificity upstream and downstream PCR primer.Perhaps, capable of using such as ClustalW (http://www.ebi.ac.uk/clustalw/) program with to there being dna sequence dna difference but keep enough specificitys and the Auele Specific Primer of the zone design of the correct amplicon of guaranteeing to increase is compared each allelic dna sequence dna.Pcr amplification of preferred design has restriction enzyme site in an allelotrope, and in other allelotrope, does not have.Long usually 18-25 the base pair of primer has similar melting temperature(Tm).

Pcr amplification

The description that PCR reaction is formed be seen everywhere (" clinical application of PCR " (ClinicalApplications of PCR), Dennis Lo (volume), Blackwell Publishing, 1998).In brief, this reaction contains primer, DNA, damping fluid and thermostability polysaccharase.Be reflected at thermal cycler, for example on the PTC-96V type MJ Research thermal cycler, the temperature step of extending through sex change, hybridization and DNA circulate (maximum 50 times).

DNA analysis

Can in all sorts of ways, comprise that carrying out molecular size with mass spectrum, capillary gel electrophoresis and agarose gel electrophoresis differentiates and analyze the PCR product.If pcr amplification of design contains different restriction enzyme sites, the DNA in available DNA-column or the deposition and purification PCR reaction, water is resuspended, carries out restricted cutting with suitable Restriction Enzyme then.DNA with restricted cutting makes agarose gel electrophoresis then, utilizes electric current that DNA is carried out size separation.Whether the various allelotrope bases of certain gene contain restriction site has different sizes.

Embodiment 2

The priority ordering of identification diagnosis property marker gene and these genes

Just organize with regard to 1, according to the empirical Bayes method of Lonnstedt and Speed (Lonnstedt and Speed, 2002, Statistica Sinica, 12:31-46) analyzing animal is in the difference that infects genetic expression before and after the EHV.Time point is analyzed before relatively infecting each time point of back and infecting.Utilize the multiple term of each animal effect, clinical state (infecting before and after the EHV) term fits to general linear model according to each gene.Back time property difference (posterior odds) according to all gene differential expressions between each clinical state group sorts to them.Only write down those genes that statistics significantly changes (rule of thumb property Bayes dwindle standard deviation (shrunken standard deviation) add up with t assess).Utilize the Holm of p value to proofread and correct (Holm, S., 1979, Scandinavian Journal of Statistics 6:65-70) keeps the strong control of 1 type specific inaccuracy.Before and after metainfective every day EHV being infected, show that the statistics gene that there were significant differences is tabulating.

In addition, clinical affected animal (clinical sign that confirmation has EHV to infect) is made comparisons with healthy animal; To regard animal with " reactivity virus infection " and healthy animal (animal of " reactivity virus infection " be usually included in known EHV metainfective about 7 day period) as makes comparisons and analyzes.

Table 5 has shown at these analyzes the gene of back according to p value ordering significant difference.This analyzes once more and compares according to proofreaied and correct the p value through the Holm method two groups." effect size (effectsize) " in these forms (M) represented logarithmic value, shows the multiple of comparing the genetic expression change with contrast.The negative value representative is reduced and is raised on the occasion of representative.The t statistics is a meaningful value as herein described with the p value.B statistics is a time property logarithm difference (Bayesian posterior log odds of differentialexpression) behind the differentially expressed Bayesian.

Table 6 has shown at these analyzes the gene of back according to T value ordering significant difference.Mark shows that which kind of gene raises and which kind of gene downward modulation (negative or just " difference " and t value), and " difference " shows the amplitude of reaction.This analyzes once more and compares according to proofread and correct the p value through the Holm method two groups.

Embodiment 3

Proof has the diagnosis potentiality to measuring herpesvirus infection

Main ingredient scoring (Jolliffe according to genetic expression calculating; I.T., " main ingredient analysis " (Principal components analysis), Springer-Verlag; 1986) adopt discrimination analysis (Venables and Ripley; 2002, " modern Application of statistics in S " (Modern AppliedStatistics in S) Springer) assessed the diagnosis potentiality of whole group of gene of each time point after the infection.Entire method is through cross validation.Each removal (drop) animal carries out cross validation (rather than once observing).Sensitivity and specificity are priority (uniform prior) through calculating.This may be interpreted as a kind of adjusting of dwindling of form, and these estimated values are narrowed down in the scope (reduced space) that reduces.

Utilize the other function score of debating of cross validation (discriminant function scores) to estimate the receptor operating curve.Crucial threshold value moved along the axle of discriminant function scoring calculate the receptor operating curve.Calculate two kinds of original inspectability ROC, and the method smoothing ROC of employing Lloyd (Lloyd C.J., 1998, Journal of the American Statistical Association, 93:1356-1364).The curve that calculates is used for the comparison of clinical feminine gender and clinical positive animal.Different curves were calculated in the genetic expression of every day after utilization was infected.Adopt trapezoidal rule, calculate area under the receptor operating curve, be applied to the ROC of empirical ROC and smoothing.

Available ROC sums up the diagnosis potentiality of certain test.Perfectly the ROC of diagnostic test is a sea line that is equal to 1 point through sensitivity and specificity.Area is 1 under the ROC of this perfect diagnosis (test).The ROC of useless diagnostic test is 45 degree lines through initial point.The area of this no information diagnosis (test) is 0.5.

Table 8 has shown area under sensitivity, selectivity and the ROC that organizes the sample of obtaining in the 2nd, 4,13 and 20 day 1 each young coltfoal infection back.Can know from these results, evidence suggests that to infect the 2nd and 4 day the diagnosis potentiality in back strong, and evidence suggests that seldom the diagnosis potentiality were arranged at the 13rd or the 20th day.

In addition,, group 1 young coltfoal makes comparisons between the 2nd, 4 and 6 day after having symptom and asymptomatic time point corresponding infection the 0th, 13 and 20 day period of back and infect.Fig. 1 shows the ROC that is used for comparison.ROC sensitivity and specificity relatively is respectively 1 and 0.867.Experience is respectively 0.892 and 0.952 with the area of smoothing under the ROC.These (results) are for providing strong evidence corresponding to the otherness genetic expression that has symptom EHV to infect.

In addition, utilize the selected gene of clinical infection animal to obtain ROC curve (Fig. 2).Utilize this check sensitivity and the specificity of genetic expression mark all outstanding.

In addition, utilization has the selected gene of reactivity virus infection animal to obtain ROC curve (Fig. 3).Utilize the sensitivity and the specificity of this check of genetic expression mark all outstanding.

In addition, utilize all genes of clinical infection animal to obtain ROC curve (Fig. 4).Utilize the sensitivity and the specificity of this check of genetic expression mark all good.

In addition, utilization has all genes of reactivity virus infection animal to obtain ROC curve (Fig. 5).Utilize the sensitivity and the specificity of check of genetic expression mark all good.

According to the estimated value of dwindling of whole group gene on the chip, the receptor operating curve that calculates is in this way guarded, that is, they tend to underestimate the diagnosis potentiality.In the operability diagnosis, should be able to obtain better diagnosis performance according to the inferior group of selected gene.

Table 7 provided organize 1 young coltfoal the 2nd, 4,6,13 with the hematology and the biological chemistry result that compared with the 0th day in 20 days, the significance,statistical of clinical parameter (heart rate (HR) and respiration rate (RR)) and these parameters.All detected values except PCV (hematocrit cell volume) all have significance,statistical, and these detected value most probables have reflected the associated change of herpesvirus infection.Yet these variations are nonspecific, also maybe be relevant with many other illnesss.These parameters of organizing 2 young coltfoals do not provide analog result, and they do not have significant difference, though these young coltfoals receive subclinical infection through managing in fact.

Fig. 6 is gene expression index (Log volume unit), serum EHV Ab level (450nm absorbancy), date, the fate (D-days) of group 1 young coltfoal, the figure of clinical sign.Organize 1 young coltfoal in inoculation on March 18th, 2003 (experimental infection).The variation of gene expression index was corresponding with existing of clinical sign, than the Zao 10-14 of appearance days of serum anti-EHV-1 antibody.This possibly be of practical significance to treatment and control than the Zao 10-14 of antibody test days diagnosis herpesvirus infections during clinical sign occurring especially.For example, present medicine is exactly the most effective during this period, and as far as many herpesvirus infections, the also during this period tool infectivity of animal.

Fig. 7 comprises that utilization organizes the figure of preceding 4 kinds of main ingredient genetic expressions of 1 young all genes of coltfoal.Each component is mapped according to postvaccinal fate.The main variation that but obvious major gene is expressed from these figure occurs in during 10 days behind the inoculation simplexvirus.During this also corresponding with existing of clinical sign (referring to Fig. 2).

Because group 2 young coltfoals experience uncontrolled natural infection in different time points, the EHV Infection Status that uses the data of organizing 1 young coltfoal the genetic expression classification (classifier) of each sample to be trained prediction group 2 young coltfoals as the basis.This prediction comprises the maximum back time property probability (posterior probability) of prediction and the Discrimination Functions scoring (it is the linear combination of each gene of sample) of prediction.Table 9 has shown the type (EHV infects positive and negative) of prediction and has differentiated scoring.

Utilize serum EHV-1Ab result, confirm that 4 young coltfoals (360,364,368 and 369: see table 9) all possibly receive natural infection (i.e. preceding 4 days of experiment beginning (the 0th day)) at-4 days.In these animals, clinical sign appearred respectively in young coltfoal 364 and 368 at the 0th and the 2nd day and the 2nd day.Divide time-like when we utilize this genetic method, it should be noted that some day or all 4 days these the young coltfoals between the 0th and the 4th day are confirmed infected by EHV-1 separately.This shows the diagnosis (index) that this EHV-1 gene expression characteristics can be EHV-1, no matter and whether animal shows clinical sign.Utilize serum EHV-1Ab result, confirm that young coltfoal 362 received natural infection at-1 day, this means should be able to be between the-1 day and the 6th day any moment detect characteristic.Utilize this gene sorting method, all observed positive findings (meaning " infected ") at the 0th, 2 and 6 day in this young coltfoal.But clinical sign does not appear in this animal.According to serum EHV-1Ab result, young coltfoal 375 is diagnosed infected about the 6th day.Observe positive classification results at the 2nd, 4 and 6 day, but also clinical sign do not occur.Utilize serum EHV-1Ab result, still be not sure of young coltfoal 366 until the 14th day and receive natural infection, this means that seropositive conversion will occur in the 27th or 28 day.Should note testing 2 animal at the 21st day all under fire (challenged).Therefore, this young coltfoal was a seronegativity at the 21st day.Except having the slight clinical sign at the 23rd day, young coltfoal 366 does not produce any clinical sign.These results show serum Ab level (humoral immunity) experiment attack in (the 21st day) almost be enough to watch for animals and can not produce the obvious sign of clinical disease.Therefore, when we do not observe any positive findings when using this genetic expression sorting technique experimental postvaccinal period.

Therefore, even be with or without in the presence of the clinical sign (or hematology and biochemical any remarkable change) and all can identifying the early stage natural infection of EHV-1 when group 2 young coltfoals demonstrate the preliminary classification indication that obtains of group 1 young coltfoal.

Embodiment 4

What have estimated performance respectively organizes gene

Though identify about 63 kinds of genes with diagnosis potentiality, common acceptable diagnosis performance only needs the seldom gene of number.

Table 10 shows cross validation classification results, sensitivity and the specificity that obtains according to the linear discriminant analysis that is selected from two kinds of genes in the group with diagnosis potentiality gene.The all of proposition is to produce the gene that maximum prefetch is surveyed the result to gene, and many other gene pairss produce acceptable classification results.Those skilled in the art are not difficult to identify other gene pairs.The right technology of identified gene includes, but is not limited to the forward mutability and selects (Venables W.N. and Ripley B.D.; " modern Application of statistics in S " (ModernApplied Statistics in S), the 4th edition, 2002.; Springer), best inferior group selection, reverse (backward) eliminate (Venables W.N. and Ripley B.D.; 2002, the same), progressively (stepwise) select (Venables W.N. and Ripley B.D., 2002; The same) and variable at random elimination (FigueradoM.A., " flexibility of supervised learning is sparse " (Adaptive Sparseness for SupervisedLearning)).

Table 11 shows that basis is selected from three kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of three kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to three kinds of HVI marker gene selects.

Table 12 shows that basis is selected from four kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of four kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to three kinds of HVI marker gene selects.

Table 13 shows that basis is selected from five kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of five kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to five kinds of HVI marker gene selects.

Table 14 shows that basis is selected from six kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of six kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to six kinds of HVI marker gene selects.

Table 15 shows that basis is selected from seven kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of seven kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to seven kinds of HVI marker gene selects.

Table 16 shows that basis is selected from eight kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of eight kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to eight kinds of HVI marker gene selects.

Table 17 shows that basis is selected from nine kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of nine kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to eight kinds of HVI marker gene selects.

Table 18 shows that basis is selected from ten kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of ten kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to ten kinds of HVI marker gene selects.

Table 19 shows that basis is selected from 20 kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of 20 kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to 20 kinds of HVI marker gene selects.

Table 20 shows that basis is selected from 30 kinds of genes of diagnostic bank and carries out the cross validation classification results that linear discriminant analysis obtains.20 groups of 20 kinds of genes only are provided.Those skilled in the art are not difficult to understand that can make other suitable diagnosis according to 20 kinds of HVI marker gene selects.

Other gene possibly introduced noise (decreasing the sensitivity and the specificity of cross validation) through overfitting (over-fitting).

Embodiment 5

Specific confirmation

Through only training classification and to surpassing 850 GeneChip according to testing data ^TMThis classification of big gene expression data transporting something containerized row, checked the specificity of said simplexvirus mark.Gene expression results in this DB derives from the horse sample of suffering from various diseases and illness, and said disease and illness comprise: the acute and chronic EPM of clinical inductive, herpesvirus infection, degenerative osteoarthritis, stress, the animal and the clinical normal animal of rhodococcus infection, endotoxemia (endotoxaemia), laminitis, stomach ulcer syndrome, training.

Two kinds of classification results have been obtained.The two is according to the reactivity virus infection and comparisons clinical normal horses.The former has utilized GeneChip ^TMOn all genes.The latter has only utilized those listed genes of table 1.The latter's mark can be identified all known EHV-infected animals.Also identify other horses of 25 unknown EHV states, comprise as another clinical trial part be in stress under animal, have the rhodococcus horse and infect sick 5 the young coltfoals that decrease of relevant known lung.

Utilize the genetic marker of this method and 63 kinds of genes, the specificity that from a group sample more than 850, detects herpesvirus infection is 95%, and sensitivity is 100%.

Embodiment 7

Gene ontology

For gene being divided into groups, adopt BLAST algorithm (Altschul, S.F., Gish according to function, metabolic process or cellular component; W., Miller, W., Myers; E.W. and Lipman, D.J., (1990), " the basic gopher of local sequence alignment " (Basic local alignment search tool); J.Mol.Biol., 215:403-410) gene order and GeneBank DB are made comparisons, carry out the retrieval of dna homolog property and gene ontology.According to these standards, table 21 has been listed the grouping of these genes.Some situation does not have available information (NA).Also referring to the table 1 of the sequence information that contains each gene.

The content of every piece of patent, patented claim and publication that this paper quotes is included this paper in as a reference in full.

Any reference that this paper quoted should not be construed as admits that this reference is that the application can profit " prior art ".

The purpose of entire description is the particular combination of describing the preferred embodiments of the invention rather than the present invention being limited to a certain embodiment or characteristic.Those skilled in the art should know in view of this paper content and can make various improvement and change and do not depart from the scope of the present invention the specific embodiments of demonstration.All this improvement will be included in the scope of additional claims with change.

Table 2

Table 3

The amino acid subclass

Table 4

Exemplary and preferred amino acids replaces

Table 5

Gene ordering according to the P value

Table 6

Gene ordering according to the T value

Table 7

EHV-1 group 1: the MV of clinical and hematologic parameter

The HR=heart rate, RR=respiration rate, Temp=rectal temperature, PCV=hematocrit cell volume (blood), TSP=total serum protein, Fib=fibrinogen, WCC=-white blood cell count(WBC), Neut=neutrophil, Lymphs=lymphocyte.

Table 8

Table 9

Organize the prediction classification and discriminating scoring of 2 young each sample of coltfoal

Table 10

Two kinds of genes selecting

Table 11

Select 3 kinds of genes

Claims (26)

1. a herpesvirus infection marker gene is used to prepare the purposes of test kit; Said test kit is used for through detecting individual interior specific immune response to the reactivity herpesvirus infection; Said herpesvirus infection marker gene is to detect before the serum simplexvirus specific antibody; In initial herpesvirus infection; In immune system cell, express; It is selected from following: (a) a kind of polynucleotide are following arbitrary nucleotide sequences: SEQ ID NO:a, b, d, f, g, h, 10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, or its complementary sequence; Or (b) polynucleotide of the polypeptide of arbitrary aminoacid sequence below a kind of coding the: SEQ ID NO:c, e, i, 11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114.

2. a nucleic probe that combines the expression product of herpesvirus infection marker gene is used to prepare the purposes of test kit; Said test kit is used to detect individual interior specific immune response to the reactivity herpesvirus infection; Said herpesvirus infection marker gene is to detect before the serum simplexvirus specific antibody; In initial herpesvirus infection; In immune system cell, express; It is selected from following: (a) a kind of polynucleotide; Be following arbitrary nucleotide sequence: SEQ ID NO:a, b, d, f, g, h, 10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, or its complementary sequence; Or (b) polynucleotide of the polypeptide of arbitrary aminoacid sequence below a kind of coding the: SEQ ID NO:c, e, i, 11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114.

3. test kit; It contains the herpesvirus infection marker gene; Said test kit is used for through detecting the specific immune response to reactivity herpesvirus infection in individual; Said herpesvirus infection marker gene is to detect before the serum simplexvirus specific antibody; In initial herpesvirus infection; In immune system cell, express; It is selected from following: (a) a kind of polynucleotide are following arbitrary nucleotide sequences: SEQ ID NO:a, b, d, f, g, h, 10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,34,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, or its complementary sequence; Or (b) polynucleotide of the polypeptide of arbitrary aminoacid sequence below a kind of coding the: SEQ ID NO:c, e, i, 11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114.

4. test kit; It contains the nucleic probe of the expression product that combines the herpesvirus infection marker gene; Said test kit is used to detect individual interior specific immune response to the reactivity herpesvirus infection; Said herpesvirus infection marker gene is to detect before the serum simplexvirus specific antibody; In initial herpesvirus infection; In immune system cell, express; It is selected from following: (a) a kind of polynucleotide are following arbitrary nucleotide sequences: SEQ ID NO:a, b, d, f, g, h, 10,12,13,15,17,19,21,23,24,25,26,27,29,31,33,4,35,37,38,39,41,43,45,47,49,51,53,55,57,59,61,63,65,66,67,69,71,73,75,76,77,79,81,83,84,85,87,89,91,93,94,96,98,99,100,101,102,104,106,107,108,109,111 or 113, or its complementary sequence; Or (b) polynucleotide of the polypeptide of arbitrary aminoacid sequence below a kind of coding the: SEQ ID NO:c, e, i, 11,14,16,18,20,22,28,30,32,36,40,42,44,46,48,50,52,56,58,60,62,64,68,70,72,74,78,80,82,86,88,90,92,95,97,103,105,110,112 or 114.

5. according to claim 1 or claim 2 purposes is characterized in that said herpesvirus infection marker gene is available from biological sample.

6. according to claim 1 or claim 2 purposes is characterized in that said biological sample comprises blood.

7. according to claim 1 or claim 2 purposes is characterized in that said biological sample comprises peripheral blood.

8. according to claim 1 or claim 2 purposes is characterized in that said biological sample comprises white corpuscle.

9. purposes as claimed in claim 2 is characterized in that, said expression product is the RNA molecule.

10. purposes as claimed in claim 2 is characterized in that, said nucleic probe is fixed on solid or the semi-solid upholder.

11. purposes as claimed in claim 2 is characterized in that, said nucleic probe constitutes the part of all nucleic probe space arrays.

12. purposes as claimed in claim 2 is characterized in that, said herpesvirus infection marker gene is the RNA molecule, detects the level that copies the bonded nucleic probe with said RNA molecule or its DNA through hybridization.

13. purposes as claimed in claim 2 is characterized in that, said herpesvirus infection marker gene is the RNA molecule, detects the level that copies the bonded nucleic probe with said RNA molecule or its DNA through nucleic acid amplification.

14. purposes as claimed in claim 2 is characterized in that, said herpesvirus infection marker gene is the RNA molecule, detects the level that copies the bonded nucleic probe with said RNA molecule or its DNA through the nucleicacidase protection test.

15. purposes as claimed in claim 2 is characterized in that, the probe that is used to detect herpesvirus infection mark polynucleotide is like the sequence shown in SEQ ID NO:145-2150 is any.

16., it is characterized in that said herpesvirus infection marker gene is available from biological sample like claim 3 or 4 described test kits.

17., it is characterized in that said biological sample comprises blood like claim 3 or 4 described test kits.

18., it is characterized in that said biological sample comprises peripheral blood like claim 3 or 4 described test kits.

19., it is characterized in that said biological sample comprises white corpuscle like claim 3 or 4 described test kits.

20. test kit as claimed in claim 4 is characterized in that, said expression product is the RNA molecule.

21. test kit as claimed in claim 4 is characterized in that, said nucleic probe is fixed on solid or the semi-solid upholder.

22. test kit as claimed in claim 4 is characterized in that, said nucleic probe constitutes the part of all nucleic probe space arrays.

23. test kit as claimed in claim 4 is characterized in that, said herpesvirus infection marker gene is the RNA molecule, detects the level that copies the bonded nucleic probe with said RNA molecule or its DNA through hybridization.

24. test kit as claimed in claim 4 is characterized in that, said herpesvirus infection marker gene is the RNA molecule, detects the level that copies the bonded nucleic probe with said RNA molecule or its DNA through nucleic acid amplification.

25. test kit as claimed in claim 4 is characterized in that, said herpesvirus infection marker gene is the RNA molecule, detects the level that copies the bonded nucleic probe with said RNA molecule or its DNA through the nucleicacidase protection test.

26. test kit as claimed in claim 4 is characterized in that, the probe that is used to detect herpesvirus infection mark polynucleotide is like the sequence shown in SEQ ID NO:145-2150 is any.

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