CN103374550A - Construction and application of SHIV (simian/human immunodeficiency virus) infectious clone - Google Patents

Abstract

The invention relates to construction and application of SHIV (simian/human immunodeficiency virus) infectious clone. Firstly, the overall length genome of a virulent strain SHIV-KB9 embedded with a B subtype env gene is cloned to a low copy number carrier pBR322, the obtained plasmid SHIV-KB9/pBR322 is taken as a genome skeleton, the env gene of Chinese main prevalent strain CRF08_BC of HIV-1 is used for substituting for corresponding gene segments so as to obtain the infectious clone SHIV-KBQJ-12, the replacing env gene is from the infectious clone strain QJ001 of HIV-1CRF08_BC and comprises the whole intracellular region, a transmembrane region and part of an extracellular region of gp120 and gp41, and the position of the replacing env gene, corresponding to the position of the nucleotide of a standard reference strain HXB2, is 6103-8249. The construction is simple and convenient to operate, has high success rate, can obtain the infectious clone in a short time, and can be used for establishing an SHIV/Chinese rhesus infection model.

Description

A kind of structure of SHIV infections clone and application thereof

Technical field

The present invention relates to the gene recombination technology field, be specifically related to the chimeric Chinese's immune deficiency virus I type of a strain (Human immunodeficiency virus-1, HIV-1) the Novel chimeric monkey/human immunodeficiency virus of Major Epidemic strain CRF08_BC env gene (Chimeric simian/human immunodeficiency virus, SHIV) construction process of infections clone also relates to simultaneously and utilizes this SHIV infections clone to set up SHIV/ rhesus monkey infection model.

The present invention is based on the achievement in research of the infective molecule cloning QJ001 of the molecular cloning strain SHIV-KB9 of widely used highly pathogenic virulent strain SHIV-89.6P in the world and the HIV-1CRF08_BC that this laboratory makes up.Wherein, the 3 '-SHIV-KB9 and 5 ' that is provided by HIV/AIDS Reagent Program-SHIV-KB9 plasmid can amplify the full-length gene group of SHIV-KB9; Infective molecule cloning QJ001 is stored among the intestinal bacteria STBL3, this intestinal bacteria called after STBL3/QJ001(Escherichia coli STBL3/QJ001), by Chinese Typical Representative culture collection center preservation (address: China, Wuhan University's Life Science College, No. 299, wuchang, wuhan district Bayi Road, 430072), preservation date is on March 5th, 2013, and preserving number is CCTCC NO:M2013066; The present invention makes up gained infective molecule cloning SHIV-KBQJ-12 and is stored among the intestinal bacteria STBL3, this intestinal bacteria called after STBL3/SHIV-KBQJ-12(Escherichia coli STBL3/SHIV-KBQJ-12), by Chinese Typical Representative culture collection center preservation (address: China, Wuhan University's Life Science College, No. 299, wuchang, wuhan district Bayi Road, 430072), preservation date is on March 5th, 2013, and preserving number is CCTCC NO:M2013067.

Background technology

Human immunodeficiency virus (Human immunodeficiency virus, HIV) infect acquired immune deficiency syndrome (AIDS) (the Acquired immunodeficiency syndrome that causes, AIDS), because its rate of propagation is fast, mortality ratio is high, now become the problem that threatens global public health maximum.

Developing safe, effective, inexpensive HIV vaccine is the most effective means of containment spread of aids, but before the HIV vaccine is carried out clinical study, need to be by test on a large scale to estimate security, validity and the immunization strategy of vaccine at animal model.Lacking at present desirable HIV vaccine, to estimate animal model be one of principal element of making a breakthrough of restriction research vaccine.Therefore, we estimate security and the validity of vaccine in the urgent need to making up a desirable animal model, and the combination of vaccine is selected, and immunization strategy is optimized.

Because HIV-1 only can infect the human and chimpanzee, and chimpanzee is animal very in imminent danger, it is limited and expensive to originate, can not use in a large number, and the carrying capacity level that HIV-1 virus copies in the chimpanzee body is very low, does not also show any disease symptoms, therefore is not suitable as laboratory animal; Once studied persons thought to study that HIV causes a disease and simian immunodeficiency virus (the Simian immunodeficiency virus of prevention mechanism " golden standard ", SIV)/the rhesus monkey model, because SIV and HIV homology on Nucleotide and amino acid levels are relatively poor, and the two serological cross reaction is limited, therefore, use this model to have very large limitation.The AIDS primate model that is most widely used at present is SHIV/ rhesus monkey model.

SHIV, namely the SIV/HIV embedded virus is to utilize gene recombination technology, the corresponding gene of SIV and HIV is replaced and the restructuring embedded virus that builds.Its biological characteristics has the characteristic of parent plant HIV and SIV concurrently, so the monkey AIDS model that brings out behind its inoculation rhesus monkey is more near human AIDS.

Traditional SHIV rejects its env, tat and rev gene at the SIVmac239 gene framework that causes a disease, and uses the corresponding gene fragment of HIV-1 to substitute and the recombinant virus of structure.SHIV virus can infect the primates such as rhesus monkey effectively, and the pathogenic SHIV virulent strain that obtains after repeatedly going down to posterity can induce infected animal that the AIDS disease occurs at short notice.SHIV/ rhesus monkey model is in the infectivity of research HIV-1 envelope protein (envelope) and pathogenic and can play a significant role aspect the AIDS vaccine immunity evaluation of HIV-1 envelope protein design; Also be good instrument at aspects such as the use of research specific cell tropism, accessory receptor and immunoreactive main target sites.

Because there is very big-difference in each hypotype strain env envelope protein gene of HIV, therefore need to make up a series of SHIV that carry HIV-1 different subtype, different strain env envelope protein genes.The envelope protein gene that the SHIV that successfully constructs at present mainly utilizes derives from HIV B hypotype strain.Wherein, the most representative is highly pathogenic virulent strain SHIV-89.6P and SHIV-KU1.SHIV-KB9 is the molecular cloning strain of SHIV-89.6P, is widely used in the world, can cause CD4 behind the inoculation rhesus monkey ⁺Lymphocyte is exhausted fast and the AIDS disease is occured, and is used as making up the genome skeleton of New type of S HIV in this patent.

Yet the B hypotype does not account for leading in the world, only accounts for less than 10% in global the infected.Therefore, the SHIV virus of extensively utilizing at present can not reflect the EPDML hereditary difference of HIV-1.Venereal Disease AIDS Preventing Controlling Center China Disease Preventing Con (NCAIDS) has carried out the large-scale HIV Molecule Epidemiology Investigation of secondary in China respectively at 1996 and 2002, and the result shows: the popular HIV-1 strain of China mainly is B ', CRF_BC and CRF_AE hypotype at present.And 1999 to 2002 during this period of time in, CRF_BC hypotype proportion amplification is larger, has leapt to the first during by 2002, reaches about 50.2%.CRF_BC is the new popular recombinant strain that B ' hypotype (Thailand B) and the restructuring of C hypotype produce, so although the env gene of the env gene of CRF_BC and C hypotype height homology is not just the same yet.CRF_BC comprises CRF07_BC hypotype and CRF08_BC hypotype, is Chinese distinctive hypotype, may obtain certain propagation advantage and the popular trend of HIV of accelerating is arranged.But so far, 4 strains are only arranged based on the gene constructed SHIV strain of C hypotype env, comprise SHIV-CHN19, SHIV-MJ4, SHIV-MCGP1.3 and SHIV-1157ipd3N4, and 3 strains comprise SHIV-XJ02170, SHIV-XJDC6431 and SHIV-CN97001 based on the gene constructed SHIV strain of CRF07_BC env; But these strain major parts all can not cause monkey AIDS disease in the monkey body, and only only having SHIV-1157ipd3N4 is present unique pathogenic virulent strain of a strain C hypotype.And, so far still have no the relevant report of any chimeric CRF08_BC env gene SHIV strain, therefore consider with the propagation advantage that this strain shows for CRF08_BC shared ratio in China the infected, we are in the urgent need to making up a chimeric New type of S HIV infective molecule cloning of Chinese HIV Major Epidemic strain CRF08_BC env gene based on the HIV-1CRF08_BC hypotype, thereby set up the SHIV/ rhesus monkey model that designs based on Chinese HIV epidemic strain and utilize this model that acquired immune deficiency syndrome (AIDS) candidate vaccine and medicine are estimated, these preventing and controlling for China's AIDS have the comparison profound significance.

In addition, because the general early detection that only can infect at HIV-1 is to the existence of the strain of CCR5 preferendum, and could detect gradually the strain of two preferendums and CXCR4 preferendum along with the progress of the acquired immune deficiency syndrome (AIDS) course of disease.And at present in the world great majority have HIV-1 strain (such as NL432) or CXCR4 and the amphitropic HIV-1 strain of CCR5 (such as 89.6) that env gene that pathogenic SHIV embedded virus utilizes all derives from the CXCR4 preferendum, this pair of preferendum HIV-1 strain also mainly utilizes CXCR4 as accessory receptor.Can cause CD4 behind these SHIV virus infection host cells ⁺Lymphocytic continuous decrease shows the feature that more HIV-1 infects late period, can not simulate well the dynamic change of the caused host's Lymphocyte depletion of HIV-1 natural infection.Therefore, it also is necessary making up a SHIV strain with CCR5 preferendum, can simulate better the feature of HIV-1 early infection like this, and its in and the resistibility of antibody also can be used for evaluation to the aids prevention vaccine.

Summary of the invention

The objective of the invention is to be to provide a kind of simple method to make up embedding and derive from the New type of S HIV plasmid of various HIV strain env genes, and can obtain rapidly at short notice new SHIV virus, thereby can analyze this viral biological characteristics.

Another object of the present invention is to be to provide a chimeric New type of S HIV infective molecule cloning of Chinese HIV-1 Major Epidemic strain CRF08_BC env gene, and utilizes the SHIV virus that obtains behind this infective molecule cloning transfection 293T cell tentatively to set up SHIV/ rhesus monkey model.This former generation SHIV virus is carried out the continuity experiment of going down to posterity in the rhesus monkey body, can further improve its virulence and pathogenic, be expected to obtain the pathogenic virulent strain of a plant height, can be used for the mechanism of causing a disease of research HIV-1 and AIDS vaccine and medicine for the design of HIV-1 envelope protein are carried out immunity evaluation.

For reaching above purpose, the present invention adopts following technical scheme:

The structure of SHIV infections clone provided by the invention, its main method is: this SHIV infections clone SHIV-KBQJ-12 the is chimeric env gene of Chinese HIV-1 Major Epidemic strain CRF08_BC, it derives from the infections clone strain QJ001 of HIV-1 CRF08_BC, the whole intracellular region, cross-film district and the part extracellular region that comprise gp120 and gp41, the position of replaced env gene of entering is 6103-8249 corresponding to the nucleotide position of canonical reference strain HXB2; The full-length gene group sequence of QJ001 is carried out evolutionary analysis show that QJ001 belongs to the CRF08_BC hypotype, equally the env gene that is entered SHIV-KBQJ-12 by new displacement is carried out evolutionary analysis and show that SHIV-KBQJ-12 belongs to the HIV-1CRF08_BC hypotype;

Described infective molecule cloning QJ001(Escherichia coli STBL3/QJ001) preserving number is CCTCC NO:M2013066, described infective molecule cloning SHIV-KBQJ-12(Escherichia coli STBL3/SHIV-KBQJ-12) preserving number is CCTCC NO:M2013067.

The present invention can adopt and comprise the steps to make up described SHIV-KBQJ-12 infections clone:

(1) employing has the plasmid pBR322 of low copy number as carrier, utilize 3 '-SHIV-KB9 and 5 ' that HIV/AIDS Reagent Program provides-SHIV-KB9 plasmid amplification to go out the full-length gene group of SHIV-KB9, and connect in the pBR322 carrier, obtain in STBL3E.coli, stablizing the SHIV-KB9/pBR322 infective molecule cloning that copies, and with this genome skeleton as structure SHIV-KBQJ-12;

(2) utilize overlap extension pcr, take SHIV-KB9/pBR322 and HIV-1QJ001 as template, increase and enzyme cuts back to close and obtains Sph I-Pst I and Pst I-two gene fragments of Xho I with corresponding primer; Again Sph I-Pst I is connected into and obtain subclone pKBQJ_SPs among the carrier pGEM5Zf (-), Pst I-Xho I is connected into obtain subclone pKBQJ_PsX among the carrier pBluescriptSK (-); The grand pKBQJ_SPs in the Asia that will obtain at last and pKBQJ_PsX are respectively after sequence verification is correct, with SphI and PstI, PstI and XhoI, SphI and XhoI difference double digestion pKBQJ_SPs, pKBQJ_PsX and SHIV-KB9/pBR322, glue connects, transforms the correct mono-clonal of STBL3E.coli picking after reclaiming required gene fragment can obtain described SHIV-KBQJ-12.

When making up infective molecule cloning SHIV-KB9/pBR322, the present invention can adopt following step:

(1) take carrier pBR322 as template, utilize primer EE_EcoA and primer EE_Eag_B to carry out pcr amplification, the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min; Then sepharose reclaims pcr amplification product, with these two enzymes of EcoRI and EagI the gene product of recovery is carried out behind the double digestion agarose gel electrophoresis again and separates enzyme and cut product;

(2) take plasmid 5 '-SHIV-KB9 as template, utilize primer KB_U5Eco_A and primer KB_pbs_B to carry out the PCR amplification, the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min; Then sepharose reclaims pcr amplification product, with these two enzymes of EcoRI and NarI the gene product of recovery is carried out behind the double digestion agarose gel electrophoresis again and separates enzyme and cut product;

(3) plasmid 5 '-SHIV-KB9 is carried out double digestion with NarI and SphI after, separate the gene fragment that enzyme is cut the about 5.6Kb of product by agarose gel electrophoresis;

Equally, plasmid 3 '-SHIV-KB9 carried out double digestion with SphI and NotI after, separate the gene fragment that enzyme is cut the about 4Kb of product by agarose gel electrophoresis;

(4) four gene fragments with above-mentioned recovery connect, and transform the STBL3E.coli competent cell, can correctly be cloned SHIV-KB9/pBR322;

Wherein, the sequence of primer is:

EE_EcoA:5’-TGT GAA TTC GCA AAC CAA CCC TTG GCA G-3’

EE_Eag_B:5’-AAA CGG CCG CGT GAT ACG CCT ATT-3’

KB_U5Eco_A:5’-AGC GAA TTC TAG ACA TAT ACT TAG AAA AGG AAG-3’

KB_pbs_B:5’-TGT TCA GGC GCC AAT CTG CTA G-3’。

The present invention can adopt following steps to make up the SHIV-KBQJ-12 infective molecule cloning after obtaining genome skeleton SHIV-KB9/pBR322:

(1) for obtaining the SphI-PstI fragment, at first take SHIV-KB9/pBR322 as template, carry out first round pcr amplification with primer tatSph_A and envSU_B, this pcr amplification reaction condition is 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take QJ001 as template, carry out first round pcr amplification with primer vpuPac_A and envCS_B simultaneously, this pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Then take two pcr amplification products of the first round as template, carry out second with primer tatSph_A and envCS_B and take turns pcr amplification, second takes turns the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take turns amplified production with SphI and PstI double digestion second again, and connect among the carrier pGEM5Zf (-), obtain subclone pKBQJ_SPs;

(2) for obtaining the PstI-XhoI fragment, at first take SHIV-KB9/pBR322 as template, carry out first round pcr amplification with primer envECD_A and SIVnef_B, this pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take QJ001 as template, carry out first round pcr amplification with primer envCS_A and envECD_B simultaneously, this pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Then take two pcr amplification products of the first round as template, carry out second with primer envCS_A and SIVnef_B and take turns amplification, second takes turns the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take turns amplified production with PstI and XhoI double digestion second again, and connect among the carrier pBluescriptSK (-), obtain subclone pKBQJ_PsX;

(3) with the subclone pKBQJ_SPs that obtains and pKBQJ_PsX respectively after sequence verification is correct, with SphI and PstI, PstI and XhoI, SphI and XhoI difference double digestion pKBQJ_SPs, pKBQJ_PsX and SHIV-KB9/pBR322, glue connects and conversion STBL3E.coli competent cell after reclaiming required gene fragment, can correctly be cloned SHIV-KBQJ-12;

Wherein, the sequence of primer is:

tatSph_A:5’-TTG GCA TGC TGT AGA GCA AGA AAT GGA GCC AGT AG-3’

envSU_B:5’-CAG GTA CCC CAT AAT AGA CTG-3’

vpuPac_A:5’-TGG TTA ATT AAA AGA ATT AGG GAA AGA GCA G-3’

envCS_B:5’-GCC CAT AGT GCT TCC TGC TGC TCC-3’

envECD_A:5’-ATT CAT AAT GAT AGT AGG AGG-3’

SIVnef_B:5’-AAG AGT CAC TGT CGC AGA TCT CC-3’

envCS_A:5’-ATA TGA GGG ACA ATT GGA GAA GTG-3’

envECD_B:5’-AAA GGT GAG TAT CCC TGC CTA ACT C-3’。

When the present invention prepares SHIV-KBQJ-12 virus, can use the Lipofectamine2000 transfection reagent that nucleotide sequence is confirmed errorless SHIV-KBQJ-12 plasmid transfection 293T cell, collect viral supernatant after 48 hours in transfection and get final product.

The present invention with SHIV-KBQJ-12 virus transfection 293T clone after, collecting cell is made ultrathin section(ing), under transmission electron microscope, can observe SHIV-KBQJ-12 and in the 293T cell, successfully assemble out complete virion, show each gene of SHIV-KBQJ-12 embedded virus succeeded express and display function.

The present invention can utilize GHOST-CD4-CXCR4 and two kinds of clones of GHOST-CD4-CCR5, detect the cell quantity of the expression GFP that is excited in the Ghost cell of expressing different accessory receptors by flow cytometer, thereby determine SHIV-KBQJ-12 virus and be with CCR5 as accessory receptor, have the characteristic of scavenger cell preferendum.

The present invention can adopt following detection method to judge the replication of SHIV-KBQJ-12 virus in people or rhesus monkey lymphocyte:

With 500ng SHIV-KBQJ-12 virus infection with PHA stimulate 72 hours 4 * 10 ⁶The PBMC cell of people or rhesus monkey, 37 ℃, 5%CO ₂Overnight incubation, removed viral supernatant liquor at the 0th day, and with the 1640 culture medium culturing cells that contain 25U/ml IL-2 and 10% foetal calf serum, then infecting the 3rd, 6,9,12,15,18,21 day, collect the supernatant sample, detect the use of SIV p27 antigen in-80 ℃ of frozen preparing against after the packing, and the adjustment cell concn is 2 * 10 ⁶Individual/ml, continue to cultivate with 1640 substratum that contain 25U/ml IL-2 and 10% foetal calf serum;

Draw by the p27 expressing quantity that is determined at the supernatant sample that above-mentioned different time points collects and to infect curve, can learn that SHIV-KBQJ-12 virus can both carry out copying of toxigenicity in the PBMC of people and rhesus monkey cell.

The present invention can adopt following detection method to judge the biological characteristics of SHIV-KBQJ-12 virus in the rhesus monkey body:

The SHIV-KBQJ-12 virus of 6000ng is inoculated two Health China rhesus monkeies in the intravenous mode of hind leg, and then gather rhesus monkey vein EDTA anticoagulation by following time point: first month gathers weekly 2 times, gathers once weekly afterwards; With above-mentioned extracting genome DNA in the rhesus monkey vein EDTA anticoagulation that different time points gathers out, and be that the specific fragment of 477bp increases to the Gag section length, to confirm whether whether SHIV-KBQJ-12 virus has been incorporated in the rhesus monkey genome, set up productive infection in the monkey body;

Behind the virus inoculation 98 days, above-mentioned two rhesus monkeies are practised mercy killing, gather various tissues, extract DNA and RNA in each tissue, Gag section length with RNA and DNA in each tissue of method detection of pcr amplification is the specific fragment of 477bp, with the infectivity of further confirmation SHIV-KBQJ-12 virus to rhesus monkey;

The result shows that above-mentioned two rhesus monkeies have begun namely to have set up stable productive infection at the 6th day and the 12nd day respectively, and viral genome also is incorporated in the rhesus monkey genome; SHIV-KBQJ-12 virus mainly is distributed in reproductive system, central nervous system and unifies in the gut associated lymphoid tissue.

Above-mentioned SHIV-KBQJ-12 infections clone provided by the invention, it is used for setting up SHIV/ China rhesus monkey infection model.

The present invention compared with prior art has following major advantage:

(1) the SHIV-KBQJ-12 infections clone that makes up of the present invention the is chimeric env gene of Chinese HIV-1 Major Epidemic strain CRF08_BC, this is the SHIV strain of the chimeric CRF08_BC env of the unique strain gene up to the present reported, and the virus of utilizing its acquisition Healthy People and rhesus monkey PBMC in preferably replication is all arranged, in the Chinese rhesus monkey body of health, also can set up stable productive infection, tentatively set up SHIV-KBQJ-12/ China rhesus monkey model, can be used for validity and security for the AIDS candidate vaccine of CRF08_BC env gene design are estimated.

(2) since infections clone in env biology of gene function determine, and from most of env gene that clinical strain increases all be do not have bioactive; And the env gene source that the SHIV-KBQJ-12 infections clone utilizes is different from the clinical strain of HIV-1 that utilized in the former bibliographical information in the infections clone of HIV-1 CRF08_BC, and the success ratio that therefore obtains New type of S HIV is significantly improved.

(3) the present invention has overcome the difficulty of the plasmid instability that comprises SHIV full-length gene group, SHIV full-length gene group is cloned on the pBR322 carrier, and to adopt STBL3E.coli be Host Strains, under 30 ℃, the specific culture condition of 180rpm, can substantially keep the stability of plasmid.Experimental result shows, utilize the STBL3E.coli Host Strains that plasmid SHIV-KB9/pBR322 and SHIV-KBQJ12/pBR322 are increased in a large number after, any disappearance does not occur in the long terminal repeat of the two.

(4) the inventive method is easy and simple to handle, and success ratio is high, and difficulty is less, can successfully make up the New type of S HIV plasmid that embeds different HIV-1 strain env gene and also can obtain rapidly at short notice new SHIV virus and then analyze this viral biological characteristics.This experiment, from amplification env gene fragment to transfectional cell after results virus only need week age, can improve significantly conventional efficient.

Description of drawings

Fig. 1 is the evolutionary analysis of HIV-1QJ001 full-length gene order

Fig. 2 is the plasmid map of SHIV-KB9/pBR322.

Fig. 3 is the structure iron of SHIV-KBQJ-12/pBR322.

Fig. 4 is the evaluation collection of illustrative plates of Sph I and Avr II double digestion plasmid SHIV-KB9/pBR322 and SHIV-KBQJ/pBR322.

In Fig. 4,0: plasmid SHIV-KB9/pBR322 double digestion after product; 1-16: 16 SHIV-KBQJ/pBR322 clones' of picking double digestion after product; Wherein, 12 SHIV-KBQJ-12/pBR322 that are among the present invention; M: maximum molecular weight is the DNA Marker of 15000bp.

Fig. 5 is the evolutionary analysis of the env gene order (HXB2:6103-8249) of SHIV-KBQJ-12/pBR322:

Fig. 6 is the electromicroscopic photograph behind the SHIV-KBQJ-12/pBR322 transfection 293T cell.

Fig. 7 is the situation that copies of SHIV-KBQJ-12 virus infection Healthy People PBMC.

Fig. 8 is the situation that copies of the healthy rhesus monkey PBMC of SHIV-KBQJ-12 virus infection.

Fig. 9 is that No. 3 rhesus monkeies inoculate after the former generation SHIV-KBQJ-12 virus DNA pcr amplification figure in the different time points whole blood.

In Fig. 9, M: maximum molecular weight is the DNA Marker of 2000bp; 1: positive control; 2: negative control; 3: behind the virus infection the 0th day; 4: behind the virus infection the 3rd day; 5: behind the virus infection the 6th day; 6: behind the virus infection the 9th day; 7: behind the virus infection the 12nd day; 8: behind the virus infection the 16th day; 9: behind the virus infection the 19th day.

Figure 10 is that No. 4 rhesus monkeies inoculate after the former generation SHIV-KBQJ-12 virus DNA pcr amplification figure in the different time points whole blood.

In Figure 10, M: maximum molecular weight is the DNA Marker of 2000bp; 1: positive control; 2: negative control; 3: behind the virus infection the 0th day; 4: behind the virus infection the 3rd day; 5: behind the virus infection the 6th day; 6: behind the virus infection the 9th day; 7: behind the virus infection the 12nd day; 8: behind the virus infection the 16th day; 9: behind the virus infection the 19th day.

Embodiment

The structure of SHIV infections clone provided by the invention and application thereof, at first will be in the world during structure the full-length gene group of the highly pathogenic virulent strain SHIV-KB9 of widely used chimeric B hypotype env gene be cloned on the low copy number carrier pBR322, obtain the higher plasmid SHIV-KB9/pBR322 of stability.Then take SHIV-KB9/pBR322 as the genome skeleton, replace with its corresponding gene fragment of env gene pairs of Chinese HIV-1 Major Epidemic strain CRF08_BC, obtain the infections clone SHIV-KBQJ-12 of chimeric CRF08_BC env gene.Wherein the new displacement env gene source of entering is in the infections clone strain QJ001 of HIV-1 CRF08_BC, the whole intracellular region, cross-film district and the part extracellular region that comprise gp120 and gp41, the position of replaced env gene of entering is 6103-8249 corresponding to the nucleotide position of canonical reference strain HXB2; The present invention is easy and simple to handle, success ratio is high, and difficulty is less, can obtain rapidly at short notice various new SHIV viruses and then analyze this viral biological characteristics, and can utilize SHIV virus to set up SHIV/ rhesus monkey model, with mechanism of causing a disease and the evaluation acquired immune deficiency syndrome (AIDS) candidate vaccine of research HIV.

Below in conjunction with embodiment and accompanying drawing the present invention is elaborated, but do not limit the present invention.

The structure of SHIV infections clone provided by the invention, its main method is: this SHIV infections clone SHIV-KBQJ-12 the is chimeric env gene of Chinese HIV-1 Major Epidemic strain CRF08_BC, it derives from the infections clone strain QJ001 of HIV-1 CRF08_BC, the whole intracellular region, cross-film district and the part extracellular region that comprise gp120 and gp41, the position of replaced env gene of entering is 6103-8249 corresponding to the nucleotide position of canonical reference strain HXB2; The full-length gene group sequence of QJ001 is carried out evolutionary analysis show that QJ001 belongs to the CRF08_BC hypotype, equally the env gene that is entered SHIV-KBQJ-12 by new displacement is carried out evolutionary analysis and show that SHIV-KBQJ-12 belongs to the HIV-1CRF08_BC hypotype;

Described infective molecule cloning QJ001(Escherichia coli STBL3/QJ001) preserving number is CCTCC NO:M2013066, described infective molecule cloning SHIV-KBQJ-12(Escherichia coli STBL3/SHIV-KBQJ-12) preserving number is CCTCC NO:M2013067.

The structure of SHIV infections clone provided by the invention, specifically adopt following step:

1. the full-length gene group sequence of HIV-1 infections clone QJ001 is carried out evolutionary analysis:

Since be set to change to SHIV-KBQJ-12 the env gene source in the infections clone QJ001 of HIV-1, therefore with MEGA5.0 software the full-length gene group sequence of QJ001 is carried out evolutionary analysis, corresponding Reference Strains sequence is from Los Alamos National Laboratory HIV Database(http: //hiv-web.lanl.gov/), evolutionary tree result (as shown in Figure 1) shows that QJ001 belongs to the CRF08_BC hypotype, and being different from the strain of C hypotype and CRF07_BC hypotype, this lays a good foundation for the structure of follow-up SHIV-KBQJ-12.

2. make up the SHIV-KB9/pBR322 plasmid:

The plasmid that carries SHIV full-length gene group is unstable because it contains direct repeat (5 ' LTR and 3 ' LTR).The SHIV-KB9 plasmid that is provided by HIV/AIDS Reagent Program is comprised of two plasmids, one of them plasmid 5 '-SHIV-KB9 comprises 5 ' LTR to vpr gene place part, and another plasmid 3 '-SHIV-KB9 comprises tat gene to 3 ' there is part in the LTR place.If want to utilize above-mentioned two plasmids to obtain virus; people must utilize this restriction enzyme site of Sph I between vpr gene and tat gene; enzyme makes the two connection after cutting these two plasmids respectively, then gained is connected the product direct transfection and allows cell (such as CEMx174).Because it is not closed annular that gained connects product, the transfection efficiency of adding suspension cell is lower, so very poor of the transfection efficiency that finally obtains, to such an extent as to we have to wait for one month after transfection or the longer time just can detect the generation of virus.

And the system that we set up, adopted the plasmid pBR322 with low copy number as carrier, utilize 3 '-SHIV-KB9 and 5 ' that HIV/AIDS Reagent Program provides-SHIV-KB9 plasmid amplification to go out the full-length gene group of SHIV-KB9, and connect in the pBR322 carrier, it is higher to transform the SHIV-KB9/pBR322 plasmid stability that obtains behind the STBL3E.coli, and can detect the generation of virus behind transfection 293T cell in several days.The genomic structure of SHIV-KB9/pBR322 as shown in Figure 2.

(1) take carrier pBR322 (available from Invitrogen company) as template, utilizes the increase gene fragment of its about 3Kb of primer EE_EcoA:5 '-TGT GAA TTC GCA AAC CAA CCC TTG GCA G-3 ' and primer EE_Eag_B:5 '-AAA CGG CCG CGT GAT ACG CCT ATT-3 '.Add in aseptic PCR pipe: 10 μ l5 * PrimeSTAR damping fluid (contains Mg ²⁺), 4 μ l2.5mM dNTPs, each 1 μ l of 10 μ M upstreams and downstream primer, 2 μ l template DNAs, 0.5 μ l PrimeSTAR HS archaeal dna polymerase (available from the precious biotech firm in Dalian) adds water to 50 μ l cumulative volumes.Reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Pcr amplification product carries out purifying and reclaims after 1% sepharose (containing 10ng/ml EB) electrophoresis is examined, with EcoRI and these two enzymes of EagI the gene product that reclaims is carried out double digestion again, endonuclease reaction operates with reference to the restriction enzyme specification sheets, after reaction finishes, separate enzyme by agarose gel electrophoresis and cut product.

(2) take plasmid 5 '-SHIV-KB9 as template, utilize the increase gene fragment of its about 800bp of primer KB_U5Eco_A:5 '-AGC GAA TTC TAG ACA TAT ACT TAG AAA AGG AAG-3 ' and primer KB_pbs_B:5 '-TGT TCA GGC GCC AAT CTG CTA G-3 '.Add in aseptic PCR pipe: 10 μ l5 * PrimeSTAR damping fluid (contains Mg ²⁺), 4 μ l2.5mM dNTPs, each 1 μ l of 10 μ M upstreams and downstream primer, 2 μ l template DNAs, 0.5 μ l PrimeSTAR HS archaeal dna polymerase (available from the precious biotech firm in Dalian) adds water to 50 μ l cumulative volumes.Reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Pcr amplification product carries out purifying and reclaims after 1% sepharose (containing 10ng/ml EB) electrophoresis is examined, with EcoRI and these two enzymes of NarI the gene product that reclaims is carried out double digestion again, endonuclease reaction operates with reference to the restriction enzyme specification sheets, after reaction finishes, separate enzyme by agarose gel electrophoresis and cut product.

(3) with reference to the restriction enzyme specification sheets plasmid 5 '-SHIV-KB9 is carried out double digestion with NarI and SphI, reaction separates respectively the gene fragment that enzyme is cut the about 5.6Kb of product by agarose gel electrophoresis after finishing

Equally, plasmid 3 '-SHIV-KB9 is carried out double digestion with SphI and NotI, reaction separates respectively the gene fragment that enzyme is cut the about 4Kb of product by agarose gel electrophoresis after finishing.

(4) four gene fragments with above-mentioned recovery connect, transform STBL3E.coli competent cell (available from Invitrogen company), 30 ℃ of dull and stereotyped cultivations after 18-20 hour, the picking mono-clonal is inoculated in the LB liquid nutrient medium that 5mL contains penbritin, 30 ℃, the 180rpm concussion is cultivated after about 14 hours, extract plasmid and identify each plasmid that extracts with SphI and Xho I double digestion, obtain size after enzyme is cut and clone for correct for the plasmid of 11.7Kb and two fragments of 2.6Kb, the pacing order of going forward side by side is to determine the accuracy of its sequence.

Annotate: related steps such as sepharose recovery, double digestion, connection and conversion in this part, all adopt the routine operation of this area.

3.SHIV-KBQJ-12 the structure of infective molecule cloning:

When making up the SHIV-KBQJ-12 infective molecule cloning, take the molecular cloning strain SHIV-KB9/pBR322 of the highly pathogenic virulent strain SHIV-89.6P that carried HIV-1B hypotype env gene as main skeleton, the env gene that will obtain from HIV-1CRF08_BC infections clone QJ001 amplification and its respective regions are replaced and are got final product to get SHIV-KBQJ-12.The env gene that new displacement is entered comprises whole intracellular region, cross-film district and the part extracellular region of gp120 and gp41, is 6103-8249 corresponding to the nucleotide position of canonical reference strain HXB2.The gene structure figure of SHIV-KBQJ-12 as shown in Figure 3.

Because the limitation that restriction enzyme site is selected, can not directly the SphI-XhoI gene fragment be replaced, and need to be take SHIV-KB9/pBR322 and QJ001 as template, method with nest-type PRC amplifies respectively two recombination fragment SphI-PstI and PstI-XhoI, these two fragments is connected among the SHIV-KB9/pBR322 behind SphI and XhoI double digestion again.

(1) for obtaining the SphI-PstI fragment, at first take SHIV-KB9/pBR322 as template, carry out first round pcr amplification with primer tatSph_A:TTG GCA TGC TGT AGA GCA AGA AAT GGA GCC AGT AG and envSU_B:CAG GTA CCC CAT AAT AGA CTG, use the PrimeSTAR HS archaeal dna polymerase of the precious biotech firm in Dalian during pcr amplification, operate in strict accordance with specification sheets, reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Simultaneously be respectively template with QJ001, carry out first round pcr amplification with primer vpuPac_A:TGG TTA ATT AAA AGA ATT AGG GAA AGA GCA G and envCS_B:GCC CAT AGT GCT TCC TGC TGC TCC, use the PrimeSTAR HS archaeal dna polymerase of the precious biotech firm in Dalian during pcr amplification, operate in strict accordance with specification sheets, reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Then take two pcr amplification products of the first round as template, carry out second with primer tatSph_A and envCS_B and take turns amplification, use the PrimeSTAR HS archaeal dna polymerase of the precious biotech firm in Dalian during pcr amplification, operate in strict accordance with specification sheets, reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Take turns amplified production with SphI and PstI double digestion second again, and connect among the carrier pGEM5Zf (-) (available from Promega company), obtain subclone pKBQJ_SPs.

(2) for obtaining the PstI-XhoI fragment, equally at first take SHIV-KB9/pBR322 as template, carry out first round pcr amplification with primer envECD_A:ATT CAT AAT GAT AGT AGG AGG and SIVnef_B:AAG AGT CAC TGT CGC AGA TCT CC, use the PrimeSTAR HS archaeal dna polymerase of the precious biotech firm in Dalian during pcr amplification, operate in strict accordance with specification sheets, reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Simultaneously take QJ001 as template, carry out respectively first round pcr amplification with primer envCS_A:ATA TGA GGG ACA ATT GGA GAA GTG and envECD_B:AAA GGT GAG TAT CCC TGC CTA ACT C, use the PrimeSTAR HS archaeal dna polymerase of the precious biotech firm in Dalian during pcr amplification, operate in strict accordance with specification sheets, reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Then take two pcr amplification products of the first round as template, carry out second with primer envCS_A and SIVnef_B and take turns amplification, use the PrimeSTAR HS archaeal dna polymerase of the precious biotech firm in Dalian during pcr amplification, operate in strict accordance with specification sheets, reaction conditions is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min.Take turns amplified production with PstI and XhoI double digestion second again, and connect among the carrier pBluescriptSK (-) (available from Stratagene company), obtain subclone pKBQJ_PsX.

(3) with the subclone pKBQJ_SPs that obtains and pKBQJ_PsX respectively after sequence verification is correct, with SphI and PstI, PstI and XhoI, SphI and XhoI respectively double digestion pKBQJ_SPs, pKBQJ_PsX and SHIV-KB9/pBR322, glue reclaims to connect after the required gene fragment and gets final product.To connect product and transform the STBL3E.coli competent cell, 30 ℃ of dull and stereotyped cultivations after 18-20 hour, the picking mono-clonal is inoculated in the LB liquid nutrient medium that 5mL contains penbritin, 30 ℃, the 180rpm concussion is cultivated after about 14 hours, extract plasmid and identify each plasmid that extracts with SphI and Avr II double digestion, obtain big or small plasmid for 11.2Kb and two fragments of 2.2Kb after enzyme is cut and be correct clone, the pacing order of going forward side by side is with the accuracy of definite its sequence.Electrophoresis is identified figure as shown in Figure 4.

Annotate: related steps such as sepharose recovery, double digestion, connection and conversion in this part, all adopt the routine operation of this area.

4.SHIV-KBQJ-12 a small amount of of plasmid is extracted:

Get final product according to the requirement in the specification sheets in the used plasmid extraction kit, the E.Z.N.A of OMEGA company is adopted in this laboratory ^TMEndoFee Plasmid Mini Kit I, step is as follows:

(1) will be inoculated in the STABLE3 of plasmid 5ml and contain in the antibiotic LB liquid nutrient medium of ammonia benzyl, 30 ℃, the 180rpm shaking table is cultivated 12-16h;

(2) get 5ml bacterium liquid, the centrifugal 1min of 10000rpm collects thalline under the room temperature;

(3) abandon substratum.Add 250ul solution I/RNase A mixed solution, the whirlpool concussion suspends thalline fully.The complete resuspended of thalline is very important for obtaining high yield;

(4) add 250ul solution II toward resuspended mixed solution, put upside down mixing 7-10 time gently.Avoid violent mixed pyrolysis liquid, otherwise can make the chromosomal DNA fracture, scission reaction does not surpass 5min;

(5) add 350ul solution III, and gentleness is put upside down centrifuge tube for several times to forming white flocks, room temperature, the centrifugal 10min of 12000rpm;

(6) get the collection tube that a clean HiBind post is put into a 2ml.Shift supernatant to pillar, 10000 * g under the room temperature, centrifugal 1 minute, make lysate flow through pillar fully, discard the filtrate in the collection tube; Repeating the 6th step to all mixed solutions all leaches from pillar.

(7) pillar is put into a new 2ml collection tube, added 500ul HB solution, the centrifugal 1min of 10000 * g abandons filtrate under the room temperature;

(8) pillar is reinstalled collection tube, adds 700ul DNA Wash Buffer, centrifugal by above-mentioned condition, abandon filtrate.

Attention: concentrated DNA Wash Buffer must press the prompting of label and use alcohol dilution before using.

(9) repeating step 8 once.

(10) abandon filtrate, pillar is reinstalled collection tube, the centrifugal void column 2min of 10000 * g is to dry pillar matrix under the room temperature.

Attention: do not ignore this step, this is most important to go out ethanol from pillar.

(11) pillar is contained on the clean 1.5ml centrifuge tube, adds the Endotoxin-free Elution Buffer (added amount depends on expection end product concentration) on pillar matrix of 30-50ul preheating, leave standstill 2min under the room temperature.The centrifugal 2min of 〉=10000 * g elutes DNA.

(12) concentration of spectrophotometric determination plasmid DNA ,-20 ℃ of preservations after the packing.

5.SHIV-KBQJ-12 a large amount of extractions of plasmid:

Because zooperal needs when a large amount of preparations do not contain endotoxic SHIV plasmid, adopt following methods to carry out:

With SHIV Plasmid Transformation STBL3 bacterial strain, 30 ℃ of dull and stereotyped cultivations after 18-20 hour, the picking mono-clonal is inoculated in the LB liquid nutrient medium that 3-5mL contains the ammonia benzyl, 30 ℃, the 180rpm concussion was cultivated after 12-14 hour, get the 1.5mL bacterial culture fluid and carry out that plasmid is little to be carried, enzyme cut identify errorless after, get remaining bacterial culture fluid 600 μ L and be inoculated in the LB liquid nutrient medium that 600mL contains the ammonia benzyl.30 ℃, 180rpm concussion were cultivated after 12 hours, get the 1.5mL bacterial culture fluid and again carry out little carrying, enzyme cut identify errorless after, with reference to MN(MACHEREY-NAGEL) specification sheets of company, the bacterium liquid of 1L is carried out a large amount of preparations of plasmid with NucleoBond Xtra Maxi EF Kit.Step is as follows:

(1) with bacterium liquid in 4 ℃, the centrifugal 10min of 4500-6000 * g thoroughly removes supernatant, precipitation is resuspended in the RES-EF solution that 24ml added RNase A, with pipettor piping and druming or with the vortex vibrator make precipitation fully concussion evenly, to without the appearance of precipitation agglomerate;

(2) add 24ml LYS-EF solution, gently put upside down centrifuge tube 5 times, leave standstill 5min under the room temperature (18 ℃-25 ℃);

Attention: before using LYS-EF solution, should observe whether the adularescent throw out occurs in this solution; If any, then should under 30 ℃ of-40 ℃ of conditions, heat this solution until all white depositions are dissolved fully, can use after making again the near room temperature of this solution temperature.

(3) after cracking fully, add 24ml NEU-EF solution, and immediately gently put upside down centrifuge tube 10-15 time, leave standstill 5min on ice behind the abundant mixing;

(4) with the NucleoBond Xtra pillar of 35mL EQU-EF solution equilibria with cylindrical filter membrane;

Edge along cylindrical filter membrane adds EQU-EF solution, and solution gradually by pillar, is guaranteed wetting whole cylindrical filter membrane under action of gravitation, and guarantees that cylindrical filter membrane can be not dry in the whole process.

(5) split product behind the mixing is directly poured in the good NucleoBond Xtra pillar with cylindrical filter membrane of pre-balance, under action of gravitation, filtered it.

(6) after lysate passes through pillar fully, with the FIL-EF elutriant wash-out impurity of 10ml.

Attention: should add along the edge of cylindrical filter membrane the FIL-EF elutriant, guaranteeing not having residue on the filter membrane, otherwise may reduce the output of plasmid.

(7) until elutriant under the action of gravitation whole cross posts after, discard cylindrical filter membrane.

(8) the ENDO-EF solution of adding 90ml makes it pass through NucleoBond Xtra pillar under action of gravity.

(9) the WASH-EF solution of adding 45ml makes it pass through NucleoBond Xtra pillar under action of gravity.

(10) the ELU-EF elutriant wash-out plasmid DNA of adding 15ml is collected the DNA elutriant with the centrifuge tube of 15ml or 50ml.

(11) Virahol under the adding 10.5ml room temperature, behind the abundant mixing, in 4 ℃, centrifugal 30 minutes of 15000 * g.

(12) carefully remove supernatant after, add 5ml and do not contain endotoxic 70% washing with alcohol precipitation, 15000 * g is centrifugal 5 minutes under the room temperature; Carefully discard the ethanol washing lotion, centrifuge tube is upside down in seasoning under the room temperature on the paper handkerchief, approximately 15-30 minute.

(13) add an amount of volume without endotoxic TE-EF or H ₂O-EF solution dissolving plasmid DNA;

(14) concentration of spectrophotometric determination plasmid DNA ,-20 ℃ of preservations after the packing.

6. the env gene order of SHIV-KBQJ-12 is carried out evolutionary analysis:

With above-mentioned method, the env gene that is entered SHIV-KBQJ-12 by new displacement is carried out evolutionary analysis, this env gene fragment is 6103-8249 corresponding to the nucleotide position of canonical reference strain HXB2.Evolutionary tree result (as shown in Figure 5) shows that the env gene of SHIV-KBQJ-12 belongs to the HIV-1CRF08_BC hypotype

7.293T the cultivation of cell:

The 293T passage cell is half attached cell, and growth is very fast, when cell state is good, when cultivating, will pass 1 generation (it is 3 bottles that the l bottle passes) in average 48 hours in containing the DMEM perfect medium of 10% foetal calf serum.

After cell covers with bottle, gently the substratum supernatant is sucked, mixture slaking liquid (the pancreatin: EDTA=1:3) peptic cell that adds pancreatin and EDTA, microscopically is observed, treat that cell begins to become bowlder, rapidly Digestive system is sopped up, add fresh DMEM perfect medium and gently cell is dispelled, 1 bottle of cell is divided into 3 bottles.37 ℃ of 5%C0 ₂Cultivate in the incubator.

8.SHIV plasmid transfection 293T clone:

This adopts Lipofectamine2000 transfection reagent (available from Invitrogen company) to carry out transfection, operates according to its specification sheets fully, and concrete steps are as follows:

(1) in transfection the day before yesterday, with DMEM perfect medium (not containing microbiotic) with the 293T cell with every hole～6 * 10 ⁵The concentration of individual cell spreads in 6 orifice plates, and incubated overnight makes cell density reach 90-95%.

(2) in two 1.5ml EP pipes, add respectively 250 μ l OPTI MEM serum free mediums, then dilute therein respectively 4 μ g plasmids and 10 μ l Lipofectamine2000 transfection reagents, gently incubated at room 5 minutes behind the mixing.

(3) then plasmid DNA and the Lipofectamine2000 of dilution of dilution mixed (cumulative volume is 500ul), mixing gently, room temperature is placed 20min.

(4) the 100ul mixed solution is dropwise joined in each hole of containing cell and substratum lentamente, shaking culture plate back and forth is evenly distributed transfection reagent, in 37 ℃ of 5%CO gently ₂Cultivate in the cell culture incubator.

(5) after transfection 4-6 hour, every hole is got the fresh DMEM perfect medium of 2ml and is changed liquid, in 37 ℃ of 5%CO ₂Cell culture incubator continues to be cultured to 48 hours.

(6) collect every hole supernatant, after filtering with 0.45 μ M filter, measure gained SHIV virus titer with SIV p27 antigen detection kit, and viral packing is frozen in-80 ℃.

9.SHIV-KBQJ-12 the electromicroscopic photograph of virion is observed:

SHIV-KBQJ-12 plasmid transfection 293T clone was collected the 293T cell after 24 hours.Abandon supernatant, add 2.5% glutaraldehyde of about 1.5ml in the capsule that cell is housed, rock gently, capsule was placed 15 minutes on ice; Scrape with cell cell is scraped off, change in the EP pipe together with glutaraldehyde, got final product in centrifugal 20 minutes under the 2500rpm speed.Sample send Wuhan Virology Institute,Chinan academy of Sciences's Electron Microscopy Room, uses the transmission electron microscope observing virion after making ultrathin section(ing).

The result shows that SHIV-KBQJ-12 successfully assembles out virion in the 293T cell, and this particle is spherical in shape, and the surface has the coating structure, as shown in Figure 6.This show each gene of SHIV-KBQJ-12 embedded virus succeeded express and display function.

10.SHIV viral p27 concentration determination

SIV p27ELISA detection kit (Advanced Bioscience Laboratories company) with ABL company is determined viral titre, and specification sheets operates in the reference reagent box, and concrete steps are as follows:

(1) guarantees that all reagent balances are to room temperature before the use.Should be noted that especially whether 20 * washing lotion the inside of observing in the test kit has obvious salt crystal, if having, should put it in 37 ℃ of baking ovens until salt crystal dissolves fully.

All testing samples are taken out from-80 ℃ of refrigerators, and 37 ℃ thaw stand-by.

(2) dilute SIV p27 standard substance according to following methods with the DMEM perfect medium:

1. metal cap and the rubber plug of the bottle of the bottled SIV of having p27 standard substance are removed.

2. according to the volume that indicates on the bottle, fully dissolve SIV p27 standard substance lyophilized powder with an amount of DMEM perfect medium, about 10 seconds of vortex gently turns upside down 5 times, leave standstill under the room temperature after 5 minutes and turn upside down 5 times again, the SIV p27 standard substance original liquid concentration of gained is 10ng/ml like this.

3. by method in the table 1 standard substance are diluted to 6 concentration in 62.5pg/ml to the 2000pg/ml scope.

(3) treat that toward the ELISA microwell plate every hole adds the 25ul lysis buffer in the gaging hole.

(4) every hole adds respectively SIV standard substance and all testing samples of 100ul serial dilution, and multiple hole is taked in suggestion; Should be specifically noted that, testing sample can be diluted to different multiples when being necessary and measure, to guarantee that measurement result is in the useful range (62.5pg/ml-2000pg/ml) of this mensuration.

Stay 4 holes that added lysis buffer, toward the perfect medium that wherein adds 100ul with as negative control hole.

(5) with sealed membrane sealing ELISA microwell plate, hatched under 37 ± 0.5 ℃ the condition 60 ± 2 minutes.

(6) 20 * washing lotion is become 1 * washing lotion with distilled water diluting, then wash plate 4 times with 1 good * washing lotion of dilution.When washing plate at every turn, the liquid in the every hole of sucking-off, every hole adds 300 μ l1 * washing lotions, soaks after 15 seconds, 96 orifice plates is upside down on the clean thieving paper pats, fully blot the inside liquid, especially last all over the time, must pat dry all liquid in the hole.

(7) every hole adds the 100ul coupling agent.

(8) with sealed membrane sealing ELISA microwell plate, hatched under 37 ± 0.5 ℃ the condition 60 ± 2 minutes.

(9) wash plate 4 times with step 6, last is all over patting dry all liquid in the hole.

(10) balance is to the peroxidase substrate of room temperature for every hole adding 100ul, and room temperature (19-23 ℃) was hatched 30 ± 1 minutes.

(11) every hole adds the 100ul stop buffer, measures the absorption value of each hole sample in 20 minutes at the wavelength of 450nm with microplate reader.

(12) with the corresponding typical curve that obtains with its concentration of the dilution standard substance absorption value of difference, calculate each absorption of sample by typical curve and be worth corresponding antibody titers.

11.Ghost the cultivation of serial clone:

Ghost series cell is attached cell, and adherent very tight, this Growth of Cells speed is slower.When cell covers with bottle when going down to posterity, gently the substratum supernatant is siphoned away, with pancreatin cell is all digested, centrifugal 5 minutes of 800rpm thoroughly removes pancreatin.With the DMEM perfect medium that contains 1 μ g/mL purine, 300 μ g/mL G418 and 15% foetal calf serum that cell is resuspended, dispel gently sub-bottle.

12. cell tropism is measured:

For determining the cell tropism of the new SHIV-KBQJ-12 of structure, selected the GHOST clone of expressing different accessory receptors, measure the situation that it utilizes accessory receptor, simultaneously with SHIV-KB9 as positive control.GHOST clone comprises GHOST-CD4-CXCR4 and two kinds of cells of GHOST-CD4-CCR5, and the two all expresses the CD4 acceptor, and expresses respectively CXCR4 and two kinds of accessory receptors of CCR5.Comprise in this clone genome and connected HIV LTR green fluorescent protein (GFP) reporter gene, and at HIV-1, the viral Tat albumen that SIV, SHIV etc. utilize different accessory receptors to enter cell activates reporter gene expression, thereby detects the situation of utilizing of virus auxiliary receptor.That is to say, after the cell with certain cell tropism is had same preferendum or amphitropic virus infection, can see that under fluorescent microscope this kind cell sends green light, thereby can judge the preferendum of virus.

Front 24 hours of infection with cell with every hole 2 * 10 ⁵The concentration of individual cell spreads in 24 orifice plates, spends the night with SHIV virus infection to be detected again, and culture changes and continues behind the liquid to cultivate 60 hours, the available fluorescence microscope cell situation that fluoresces.Further collecting cell is also resuspended with the PBS washing that contains 2%, collects 20,000 cells and carries out flow cytometer detection.Can analyze the preferendum of SHIV-KBQJ-12 according to the result of flow cytometer detection.

Experimental result (as shown in table 4) shows that viral SHIV-KBQJ-12 is the virus of having a liking for scavenger cell of using the CCR5 accessory receptor, and it can simulate the feature of HIV early infection better like this.

13. separation and the cultivation of human peripheral monokaryon lymphocyte (PBMC):

This used human lymphocyte parting liquid is the product of Tianjin Hao ocean biological products science and technology limited Company, and article No. LTS1077 operates according to its specification sheets fully, and concrete steps are as follows:

Gather 50 milliliters of EDTA anticoagulated whole bloods of the healthy volunteer that donates blood, carry out by the following method the separation of PBMC:

(1) with 1 * PBS the EDTA anticoagulated whole blood is diluted 2-3 doubly, gently mixing.

(2) in each 15ml centrifuge tube, add first 5ml Ficoll-Paque PLUS solution, and then add gently the blood sample of 5ml dilution, it is placed on the Ficoll-Paque PLUS layer.In the time of should paying special attention to add dilute blood, make sample and Ficoll-Paque PLUS layering, the two must not mix as far as possible.

(3) under 18-20 ℃ of temperature, with centrifugal 15 minutes of 1500 rev/mins of rotating speeds (revolutions per minute, rpm).Should be specifically noted that acceleration and retarded velocity all need be arranged to minimum.

(4) take out gently centrifuge tube, as seen be followed successively by plasma layer, ring-type oyster white buffy coat, transparent separation liquid layer, red corpuscle layer from top to bottom.

Use aseptic transfer pipet to extract plasma layer liquid out, buffy coat is being maintained the original state at the interface.Should note buffy coat not being confused.The supernatant liquid that comprises blood plasma can store to be used after a while.

(5) use an aseptic transfer pipet that buffy coat (one deck tunica albuginea) is moved in the new aseptic centrifuge tube lightly.It is most important to move down out the material that the place, interface has in the situation of minimum.Removing unnecessary Ficoll-Paque PLUS can cause granulocyte to pollute; Remove unnecessary supernatant liquor and can cause that then thrombocyte pollutes.

(6) to the PBS solution that adds at least 3 times of volumes in the lymphocytic test tube is housed.

(7) with transfer pipet lightly with the cell pressure-vaccum, make its suspension.

(8) under 18-20 ℃, with 1500rpm speed centrifugal 10 minutes.

(9) abandon supernatant liquor,, make in its PBS solution that is suspended in 6-8ml lightly with the lymphocyte pressure-vaccum with transfer pipet.

(10) under 18-20 ℃, with 1000rpm speed centrifugal 5 minutes.

(11) abandon supernatant liquor, with the lymphocyte that 1640 perfect mediums suspend and separate, adjusting cell concn is 2 * 10 ⁶Cells/ml, and add PHA5ug/ml minute installs to that 37 ℃ of cultivations get final product in the culturing bottle.

14. separation and the cultivation of monkey peripheral blood mononuclear lymphocyte (PBMC):

This adopts the Ficoll-Paque PLUS of GE company that rhesus monkey PBMC is separated, and concrete steps are as follows:

Gather 50 milliliters of anticoagulated whole bloods of healthy rhesus monkey, carry out by the following method the separation of PBMC:

(1) fresh EDTA anticoagulated blood and the balanced salt solution of equivalent are mixed;

(2) in each 15ml centrifuge tube, add first 3ml Ficoll-Paque PLUS solution, add gently again the blood sample of 4ml dilution, it is placed on the Ficoll-Paque PREMIUM layer.In the time of should paying special attention to add dilute blood, make sample and Ficoll-Paque PREMIUM layering, the two must not mix as far as possible.

(3) under 18-20 ℃ of temperature, centrifugal 400 * g30-40 minute.Should be specifically noted that acceleration and retarded velocity all need be arranged to minimum.

(4) take out gently centrifuge tube, from top to bottom as seen be followed successively by plasma layer, buffy coat, separation liquid layer, red corpuscle layer.

Use aseptic transfer pipet to extract plasma layer liquid out, buffy coat is being maintained the original state at the interface.Should note buffy coat not being confused.The supernatant liquid that comprises blood plasma can store to be used after a while.

(5) use an aseptic transfer pipet that buffy coat (one deck tunica albuginea) is moved in the new aseptic centrifuge tube lightly.It is most important to move down out the material that the place, interface has in the situation of minimum.Removing unnecessary Ficoll-Paque PLUS can cause granulocyte to pollute; Remove unnecessary supernatant liquor and can cause that then thrombocyte pollutes.

(6) to the balance liquid solution that adds at least 3 times of volumes in the lymphocytic test tube is housed.

(7) with transfer pipet lightly with the cell pressure-vaccum, make its suspension.

(8) under 18-20 ℃, with 60-100 * g speed centrifugal 10 minutes.

(9) abandon supernatant liquor,, make in its balanced salt solution that is suspended in 6-8ml lightly with the lymphocyte pressure-vaccum with transfer pipet.

(10) under 18-20 ℃, with 60-100 * g speed centrifugal 10 minutes.

(11) abandon supernatant liquor, with the lymphocyte that 1640 perfect mediums suspend and separate, adjusting cell concn is 2 * 10 ⁶Cells/ml, and add PHA5ug/ml minute installs to that 37 ℃ of cultivations get final product in the culturing bottle.

Annotate: prepare balanced salt solution, the solution A (table 2) of 1 deal is mixed getting final product with the solution B (table 3) of 9 deals.

Referring to table 2, stoste A is dissolved in about 950ml distilled water, add concentrated hydrochloric acid (HCl), making its pH value is 7.6, then adjusts its volume and namely makes solution A to 1L.

15.SHIV the detection of virus replication in the human PBMC:

(1) the above-mentioned SHIV-KBQJ-12 for preparing and SHIV-KB9 virus liquid are thawed at 37 ℃ respectively.

The Healthy People PBMC that (2) will stimulate 3 days with PHA presses every pipe 4 * 10 ⁶After the individual cell packing, centrifugal 5 minutes of 1000rpm, precipitation is suspended in 1640 substratum that 0.5ml contains 10% foetal calf serum.

(3) with the SHIV-KBQJ-12 virus liquid of 500ng and SHIV-KB9 virus liquid respectively with 4 * 10 ⁶People's PBMC mixes, and supply with 1640 substratum that contain 10% foetal calf serum and respectively manage cumulative volume to 2ml, 37 ℃, 5%CO ₂Overnight incubation.

(4) 1000rpm, 4 ℃ are centrifugal 5 minutes, remove viral supernatant liquor; Wash cell with substratum, centrifugal, so repeat two times, at last precipitation is resuspended in 1640 substratum that contain 25U/ml IL-2 and 10% foetal calf serum and changes in the Tissue Culture Flask, 37 ℃, 5%CO ₂Cultivate, set up and infect, be designated as the 0th day.

(5) infecting the 3rd, 6,9,12,15,18,21 days, centrifugal 5 minutes of 1000rpm, 4 ℃ collect 2ml supernatant sample, and-80 ℃ are frozenly used in order to detecting SIV p27 antigens after the packing; Again wash cell with the substratum about 5ml, centrifugal and be resuspended in 1640 substratum that contain 25U/ml IL-2 and 10% foetal calf serum, adjusting cell concn is 2 * 10 ⁶Individual/ml.Need to replenish the PBMC of an amount of fresh stimulation in the time of necessary.

(6) will carry out at the supernatant sample that different time points is collected SIV p27 cAg and detect, and the result be added up draw out the infection curve, as shown in Figure 7.

The result shows, when infecting the human PBMC with the viral supernatant of collecting behind the transfection 293T cell, is infecting virus rear the 9th day, and it is the highest that virus titer reaches.SHIV-KB9 is up to 198ng/ml, and SHIV-KBQJ-12 is up to 187ng/ml, only a little less than pathogenic virulent strain SHIV-KB9.

16.SHIV the detection of virus replication in rhesus monkey PBMC:

(1) the above-mentioned SHIV-KBQJ-12 for preparing and SHIV-KB9 virus liquid are thawed at 37 ℃ respectively.

The Healthy People PBMC that (2) will stimulate 3 days with PHA presses every pipe 4 * 10 ⁶After the individual cell packing, centrifugal 5 minutes of 1000rpm, precipitation is suspended in 1640 substratum that 0.5ml contains 10% foetal calf serum.

(3) with the SHIV-KBQJ-12 virus liquid of 500ng and SHIV-KB9 virus liquid respectively with 4 * 10 ⁶People's PBMC mixes, and 37 ℃, 5%CO ₂Overnight incubation.

(4) 1000rpm, 4 ℃ are centrifugal 5 minutes, remove viral supernatant liquor; Wash cell with substratum, centrifugal, so repeat two times, at last precipitation is resuspended in 1640 substratum that contain 25U/ml IL-2 and 10% foetal calf serum and changes in the Tissue Culture Flask, 37 ℃, 5%CO ₂Cultivate, set up and infect, be designated as the 0th day.

(5) infecting the 3rd, 6,9,12,15,18,21 days, centrifugal 5 minutes of 1000rpm, 4 ℃ collect 2ml supernatant sample, and-80 ℃ are frozenly used in order to detecting SIV p27 antigens after the packing; Again wash cell with the substratum about 5ml, centrifugal and be resuspended in 1640 substratum that contain 25U/ml IL-2 and 10% foetal calf serum, adjusting cell concn is 2 * 10 ⁶Individual/ml.Need to replenish the PBMC of an amount of fresh stimulation in the time of necessary.

(6) will carry out at the supernatant sample that different time points is collected SIV p27 cAg and detect, and the result be added up draw out the infection curve, as shown in Figure 8.

The result shows, when infecting rhesus monkey PBMC with the viral supernatant of collecting behind the transfection 293T cell, is infecting virus rear the 12nd day, and it is the highest that virus titer reaches.SHIV-KB9 is up to 76.1ng/ml, and SHIV-KBQJ-12 is up to 18.9ng/ml.

17.SHIV-KBQJ-12 the biological characteristics of virus in the rhesus monkey body detects:

Adopt health check-up to confirm without unusual in the experiment, and through serology indirect immunofluorescence antibody test procedure inspection eliminating simian immunodeficiency virus (SIV), monkey reverse transcription D C-type virus C (SRV_1,2,5) and 2 of the rhesus monkeies (being numbered No. 3 and No. 4) that infect of monkey T lymphatic I C-type virus C (STLV_1), respectively inoculate the virus liquid that 6000ng produces after with SHIV-KBQJ-12 transfection 293T cell for the rhesus monkey of two health in the intravenous mode of hind leg, then gather rhesus monkey vein EDTA anticoagulation by following time point: first month gathers weekly twice, gathers once weekly afterwards.

This uses the QIAGEN DNeasy Blood﹠amp of company; Tissue Kit extracts the DNA in whole blood and the tissue, operates according to the specification sheets that provides in the test kit fully, and concrete steps are as follows:

(1) before the experiment whole blood or tissue samples, Proteinase K are returned to room temperature and be ready to 56 ℃ of water-baths.

(2) if whole blood sample adds 20 μ l Proteinase Ks in 1.5ml centrifuge tube bottom first, then get 200 μ l whole blood samples and add in the above-mentioned centrifuge tube, change step 4 over to behind the appropriate mixing and operate.

(3) if tissue samples, first each tissue is shredded that (spleen need be less than 10mg, other tissue need be less than 25mg) and put into the centrifuge tube of a 1.5ml, add 180 μ l ATL solution, 20 μ l Proteinase Ks, the vortex concussion is with mixing, hatch in 56 ℃ of water-baths until in a organized way fully cracking of institute, of short durationly change step 4 over to after centrifugal and operate.

(4) 200 μ l AL liquid are added in the above-mentioned centrifuge tube, fully shake mixing.

(5) above-mentioned mixed solution is placed 56 ℃ of water-baths hatched 10 minutes.

Attention: excessive temperature or after the degraded that can cause for a long time DNA.

(6) above-mentioned 1.5ml centrifuge tube is of short duration centrifugal, be bonded at drop on the tube wall with elimination.

(7) in centrifuge tube, add 200 μ l dehydrated alcohols, fully shake mixing and of short duration centrifugal.

(8) above-mentioned mixed solution is transferred to cover the centrifugal 1min of 8000rpm is arranged in the DNeasy Mini spin pillar of 2ml collection tube.The 2ml collection tube that renews.

(9) in pillar, add 500 μ l AW1 solution, the centrifugal 1min of 8000rpm.The 2ml collection tube that renews.

(10) in pillar, add 500 μ l AW2 solution, the centrifugal 3min of 14000rpm.The 2ml collection tube that renews.

(11) pillar is placed new 1.5ml centrifuge tube, the AE solution that 200 μ l is preheated to 56 ℃ joins in the pillar, hatches after 1 minute the centrifugal 1min of 10000rpm under the room temperature with eluted dna.

(12) get the DNA that 3 μ l extract and carry out agarose gel electrophoresis, and with the quality of nucleic acid that UV spectrophotometer measuring is extracted.

With behind the SHIV KBQJ12 virus injection rhesus monkey the 0th, 3,6,9,12,16, extracting genome DNA in the rhesus monkey vein EDTA anticoagulation that gathered in 19,23 and 28 days and is that the specific fragment of 477bp increases to Gag conserved regions length out, take nest-type PRC, first round amplification use following SIV out F and SIV out R this to primer:

SIV out F:5’-GGTGCATTCACGCAGAAGAGAAAGTGAAAC-3’，

SIV out R:5’-TGGGTTAGCATTTTGAATCAGCAGTGTTTG-3’，

The first round reaction conditions of amplification is: 94 ℃ of 3min, 50 ℃ of 1min, 72 ℃ of 1min; 94 ℃ of 30S, 62 ℃ of 30S ,-0.3 ℃/circulation, 72 ℃ of 1min, 34 circulations; 72 ℃ of 10min.

Second take turns amplification use following SIV inner F and SIV inner R this to primer:

SIV inner F:5’-GCAGAGACATCTAGTGGTGGAAACAGGAAC-3’

SIV inner R:5’-AATGTTGCCTACTGGTATGGGGTTTTGTTG-3’，

The second reaction conditions of taking turns amplification is: 94 ℃ of 3min, 50 ℃ of 1min, 72 ℃ of 1min; 94 ℃ of 30S, 54 ℃ of 30S, 72 ℃ of 1min, 34 circulations; 72 ℃ of 10min.Reaction is carried out agarose electrophoresis with the PCR product after finishing, and the electrophoresis result of No. 3 and No. 4 rhesus monkeies respectively as shown in Figure 9 and Figure 10.

The result shows, No. 3 and No. 4 rhesus monkeies after the 12nd day and the beginning in the 6th day, can both detect viral gag gene respectively always after infecting virus.Illustrate that SHIV-KBQJ-12 virus has been incorporated in the rhesus monkey genome, has set up stable productive infection in the monkey body.

Behind the virus inoculation 98 days, put to death above-mentioned two rhesus monkeies, gather the various tissues such as inguinal region, mesentery, with the DNeasy Blood﹠amp of QIAGEN company; Tissue Kit and RNeasy Mini Kit test kit extract respectively DNA and the RNA in each tissue, in the tissue extraction step of DNA as mentioned above, the concrete steps of extracting RNA in the tissue are as follows:

(1) tissue of getting first no more than 30mg is put into the centrifuge tube of a 1.5ml, shreds rear adding 600 μ l and has added the RLT solution of β-ME and fully grind.

(2) with maximum speed of revolution with the above-mentioned centrifugal 3min of centrifuge tube that lysate is housed, carefully take out in supernatant to the clean new centrifuge tube.

(3) 70% ethanol that adds equivalent volumes in above-mentioned supernatant liquor, rapid mixing.

(4) above-mentioned mixed solution being transferred to cover has in the RNeasy spin pillar of 2ml collection tube, covers gently upper tube cap, and the centrifugal 15s of 12000rpm discards filtrate.

(5) add 700 μ l RW1 solution in pillar, cover gently upper tube cap, the centrifugal 15s of 12000rpm discards filtrate.

(6) add the RPE solution that 500 μ l have added ethanol in pillar, cover gently upper tube cap, the centrifugal 15s of 12000rpm discards filtrate.

(7) add the RPE solution that 500 μ l have added ethanol in the pillar, cover gently upper tube cap, the centrifugal 2min of 12000rpm changes the centrifugal 1min of 12000rpm behind the new 2ml collection tube.

(8) the 1.5ml centrifuge tube that pillar is placed new test kit provide directly joins 30-50 μ l above the film of pillar without RNA enzyme water, covers gently upper tube cap, hatches after 1 minute the centrifugal 1min of 12000rpm under the room temperature with eluted rna.

After extracting the DNA and RNA in each tissue, the Gag section length with RNA and DNA in each tissue of method detection of pcr amplification is the specific fragment of 477bp equally, and statistics is as shown in table 5, and " ND " represents undetermined in the table 5.

The result shows, reproductive system, the central nervous system that virus mainly is present in rhesus monkey unified in the gut associated lymphoid tissue, this further confirmed SHIV-KBQJ-12 virus to rhesus monkey be have infective.

Therefore, we have tentatively set up the SHIV-KBQJ-12/ rhesus monkey model of chimeric Chinese HIV-1 Major Epidemic strain CRF08_BC env gene.But because lower without the SHIV that goes down to posterity viral infectivity of former generation, what the SHIV-KBQJ-12 strain showed also is the part infection characterization that is similar to its parent plant HIV-1QJ001, and it uses the CCR5 accessory receptor, and disease process is slower.After the experiment, the replication of virus and pathogenic meeting improve greatly but process monkey body goes down to posterity.This low virulent strain that makes up at present/rhesus monkey model can be simulated the chronic disease process that generally occurs after the HIV natural infection, thereby also has certain application prospect in HIV candidate vaccine and drug screening.

Subordinate list

Table 1

Table 2

Table 3

Table 4

Table 5

＜110〉Wuhan Virology Institute,Chinan academy of Sciences

＜120〉a kind of structure of SHIV infections clone and application thereof

<140>

<141>

<160> 19

<170>

<210> 1

<211> 28

<212> DNA

＜213〉artificial primer (EE_EcoA)

<220>

<221>

<222>

<223>

<400> 1

TGT GAA TTC GCA AAC CAA CCC TTG GCA G

<210> 2

<211> 24

<212> DNA

＜213〉artificial primer (EE_Eag_B)

<220>

<221>

<222>

<223>

<400> 2

AAA CGG CCG CGT GAT ACG CCT ATT

<210> 3

<211> 33

<212> DNA

＜213〉artificial primer (KB_U5Eco_A)

<220>

<221>

<222>

<223>

<400> 3

AGC GAA TTC TAG ACA TAT ACT TAG AAA AGG AAG

<210> 4

<211> 22

<212> DNA

＜213〉artificial primer (KB_pbs_B)

<220>

<221>

<222>

<223>

<400> 4

TGT TCA GGC GCC AAT CTG CTA G

<210> 5

<211> 35

<212> DNA

＜213〉artificial primer (tatSph_A)

<220>

<221>

<222>

<223>

<400> 5

TTG GCA TGC TGT AGA GCA AGA AAT GGA GCC AGT AG

<210> 6

<211> 21

<212> DNA

＜213〉artificial primer (envSU_B)

<220>

<221>

<222>

<223>

<400> 6

CAG GTA CCC CAT AAT AGA CTG

<210> 7

<211> 31

<212> DNA

＜213〉artificial primer (vpuPac_A)

<220>

<221>

<222>

<223>

<400> 7

TGG TTA ATT AAA AGA ATT AGG GAA AGA GCA G

<210> 8

<211> 24

<212> DNA

＜213〉artificial primer (envCS_B)

<220>

<221>

<222>

<223>

<400> 8

GCC CAT AGT GCT TCC TGC TGC TCC

<210> 9

<211> 21

<212> DNA

＜213〉artificial primer (envECD_A)

<220>

<221>

<222>

<223>

<400> 9

ATT CAT AAT GAT AGT AGG AGG

<210> 10

<211> 23

<212> DNA

＜213〉artificial primer (SIVnef_B)

<220>

<221>

<222>

<223>

<400> 10

AAG AGT CAC TGT CGC AGA TCT CC

<210> 11

<211> 24

<212> DNA

＜213〉artificial primer (envCS_A)

<220>

<221>

<222>

<223>

<400> 11

ATA TGA GGG ACA ATT GGA GAA GTG

<210> 12

<211> 25

<212> DNA

＜213〉artificial primer (envECD_B)

<220>

<221>

<222>

<223>

<400> 12

AAA GGT GAG TAT CCC TGC CTA ACT C

<210> 13

<211> 30

<212> DNA

＜213〉artificial primer (SIV out F)

<220>

<221>

<222>

<223>

<400> 13

GGT GCA TTC ACG CAG AAG AGA AAG TGA AAC

<210> 14

<211> 30

<212> DNA

＜213〉artificial primer (SIV out R)

<220>

<221>

<222>

<223>

<400> 14

TGG GTT AGC ATT TTG AAT CAG CAG TGT TTG

<210> 15

<211> 30

<212> DNA

＜213〉artificial primer (SIV inner F)

<220>

<221>

<222>

<223>

<400> 15

GCA GAG ACA TCT AGT GGT GGA AAC AGG AAC

<210> 16

<211> 30

<212> DNA

＜213〉artificial primer (SIV inner R)

<220>

<221>

<222>

<223>

<400> 16

AAT GTT GCC TAC TGG TAT GGG GTT TTG TTG

<210> 17

<211> 9742bp

<212> DNA

＜213〉artificial sequence (QJ001 infections clone whole genome sequence)

<220>

<221>

<222>

<223>

<400> 17

1 TGGAAGGGTT AATTTACTCT AAGAAAAGCC AAGAGATCCT TGACTTGTGG GTCTATCACA

61 CACAAGGCTA CTTCCCTGAT TGGCAAAACT ACACGCCGGG ACCAGGGGTC AGATTCCCAC

121 TGACTTTTGG GTGGTGCTTT AAGCTAGTAC CAGTTGACCC AAGGGAAGTA GAAGAGGCCA

181 ACGAAGGAGA AGACAACTGC TTGCTACACC CTGTCTGCCA GCATGGAATG GATGATGAAC

241 ACGGAGAAGT ATTAAAGTGG AAGTTTGACA GTCAACTAGC ACGCAGACAC ATGGCCCGCG

301 AGCTACATCC GGAGTTTTAC AAAGACTGCT GACACAGAAG GGACTGTCTG CTGACACAGA

361 AGGGACTTTC CGCGGGACTT TCCACTGGGG CGTTCCGGGA GGTGTGGTCT GGGCGGGACT

421 GGGAGTGGTC AACCCTCAGA TGCTGCATAT AAGCAGCTGC TTTTCGCCTG TACTGGGTCT

481 CTCTAGTTAG ACCAGATCTG AGCCTGGGAG CTCTCTGGCT AGCTAGGGAA CCCACTGCTT

541 AAGCCTCAAT AAAGCTTGCC TTGAGTGCTC TGAGCAGTGT GTGCCCGTCT ATTGTGTGAC

601 TCTGGTAACT AGAGATCCCT CAGACCCTTG TGGTAGTGTG GAAAATCTCT AGAAGTGGCG

661 CCCGAACAGG GACTTGAAAG CGAAAGTAAG ACCAGAGGAG ATCTCTCGACGCAGGACTCG

721 GCTTGCTGAA GTGCACTCGG CAAGAGGCGA GAGCGGCGAC TGGTGAGTAC GCCAATTTTA

781 TTTGACTAGC GGAGGCTAGA AGGAGAGAGA TGGGTGCGAG AGCGTCAATACTAAGAGGGG

841 GAAAATTAGA TAAATGGGAA AAAATTAGGT TAAGGCCAGG GGGAAAGAAACACTATATGC

901 TAAAACACCT AGTATGGGCA AGCAGGGAGC TGGAAAGATT TGCACTTAAC CCTGGCCTTT

961 TAGAGACATC AGAAGGCTGT AAGCAAATAA TAAAACAGCT ACAACCAGCT CTTCAGACAG

1021 GAACAGAGGA ACTTAGATCA TTATTCAACA CAGTAGCAAC TCTCTACTGT GTACATGCAG

1081 GGATAGAAGT ACGAGACACC AAAGAAGCCT TAGACAGGAT AGAGGAAGAA CAAAAGAAAG

1141 TTCAGCAAAA AACACAGCAG ACAAAAGAGG CTGACGGGAA GGTCAGTCAA AATTATCCTA

1201 TAGTGCAGAA TCTCCAAGGG CAAATGGTAC ATCAGCCCAT ATCACCTAGA ACTTTAAATG

1261 CATGGGTAAA AGTAGTAGAA GAGAAGGCCT TTAGCCCAGA AGTGATACCC ATGTTTACAG

1321 CATTATCAGA AGGAGCCACC CCACAAGATT TAAACACCAT GTTAAATACA GTCGGGGGAC

1381 ATCAAGCAGC CATGCAAATG TTAAAAGATA CCATCAATGA AGAGGCTGCA GAATGGGATA

1441 GATTGCATCC AGTGCATGCA GGGCCAGTGG CACCAGGCCA GATGAGAGAA CCAAGGGGAA

1501 GTGACATAGC AGGAACTACT AGTACTCTTC AGGAGCAAAT AGGATGGATG ACAAATAATC

1561 CACCTGTCCC AGTAGGAGAA ATCTATAAAA GGTGGATAAT CCTGGGATTA AATAAAATAG

1621 TAAGAATGTA TAGCCCTACC AGCATTTTGG ACATAAAACA AGGGCCAAAG GAACCCTTTA

1681 GAGACTATGT AGACCGGTTC TTTAAAACTT TAAGAGCTGA ACAAGCTACA CAAGATGTAA

1741 AAAATTGGAT GACAGACACC TTGTTAGTCC AAAATGCGAA CCCAGATTGT AAGACCATTT

1801 TAAGAGCATT AGGACCAGGG GCTTCATTAG AAGAGATGAT GACAGCATGC CAGGGAGTAG

1861 GAGGGCCTAG CCACAAAGCA AGAGTGTTGG CTGAGGCAAT GAGCCAAACA AACAATACCA

1921 TAATGATGCA GAGAAGCAAT TTTAAAGGCT CTAAAAGAAT TGTTAAATGC TTCAACTGTG

1981 GCAAGGAAGG GCACATAGCC AGAAATTGCA GGGCCCCTAG GAAAAAAGGC TGTTGGAAAT

2041 GTGGAAAGGA AGGACACCAA ATGAAAGACT GTACTGAAAG GCAGGCTAAT TTTTTAGGGA

2101 AAATTTGGCC TTCCCACAAG GGGAGGCCAG GGAATTTCCT CCAGAGCAGA CCAGAGCCAA

2161 CAGCTCCACC AGCAGAGAGC TTCAGGTTCG AGGAGACAAC CCCAGCTCCG AAACAGGAAC

2221 CGAAAGACAG GGAACCCTTA ACTTCCCTCA GATCACTCTT TGGCAGCGAC CCCTTGTCTC

2281 AATAAAAGTA GGAGGCCAGA TAAAAGAGGC TCTCTTAGAC ACAGGAGCAG ATGATACAGT

2341 ATTAGAAGAG GTAAATTTGC CAGGAAAATG GAAACCGAAA ATGATAGGAG GAATTGGAGG

2401 TTTTATCAAA GTAAGACAAT ATGAGCAAAT ACCTATAGAA ATTTGTGGAA AAAAGGCTAT

2461 AGGTACAGTA TTAGTGGGAC CCACACCTGT CAACATAATT GGAAGAAATA TGTTGACCCA

2521 GCTTGGATGC ACACTAAATT TTCCAATCAG TCCCATTGAA ACTGTACCAG TAAAATTAAA

2581 GCCAGGAATG GATGGCCCAA AGGTTAAACA ATGGCCATTG ACAGAAGAAA AAATAAAAGC

2641 ATTAACAGCA ATTTGTGATG AAATGGAGAA GGAAGGAAAA ATTACAAAAA TTGGGCCTGA

2701 CAATCCATAT AACACTCCAA TATTTGCCAT AAAAAAGAAG GACAGTACTA AGTGGAGAAA

2761 ATTAGTAGAT TTCAGGGAAC TCAATAAAAG AACTCAAGAT TTTTGGGAAG TTCAATTAGG

2821 AATACCACAC CCAGCAGGGT TAAAAAAGAA AAAATCAGTA ACAGTCCTGG ATGTGGGTGA

2881 TGCATATTTC TCAGTTCCTT TAGATAAAGA CTTCAGGAAG TATACTGCAT TTACCATACC

2941 TAGTGTAAAC AATGAGACAC CAGGAATTAG ATATCAGTAC AATGTGCTTC CACAGGGATG

3001 GAAAGGATCA CCAGCAATAT TCCAATGTAG CATGACAAAA ATCTTAGAGC CTTTTAGAAA

3061 ACACAATCCA GACCTAGTTA TCTATCAATA CATGGATGAC TTGTATGTAG GATCTGACTT

3121 AGAAATAGGG CAGCATAGAA CAAAAATAGA GGAACTGAGA GAACATCTGT TAAAGTGGGG

3181 ATTTACCACA CCAGACAAGA AACATCAGAA AGAACCTCCA TTTCTATGGA TGGGGTATGA

3241 ACTCCATCCT GACAAATGGA CAGTACAGCC TATACAGCTG CCAGAAAAGG ATAGCTGGAC

3301 TGTCAATGAT ATACAGAAGT TAGTGGGAAA ATTAAACTGG GCAAGTCAGA TTTACCCAGG

3361 AATTAAAGTA AGGCAACTTT GTAAACTCCT TAGGGGGACC AAAGCACTAA CAGACATAGT

3421 ACCACTAACT GAAGAAGCAG AATTAGAATT GGCAGAAAAC AGGGAAATTT TAAAAGAACC

3481 AGTACATGGA GCATATTATG ACCCATCAAA AGAATTGATA GCTGAAATAC AGAAACAGGG

3541 GCAGGACCAA TGGACATATC AAATTTACCA AGAACCATTC AAAAACCTGA AAACAGGAAA

3601 GTATGCAAAA ATGAGGACTG CCCACACTAA TGATGTAAAA CAGTTAACAG AGGCGGTGCA

3661 AAAAATAGCC ATGGAAAGCA TAGTAATATG GGGAAAGATT CCTAAATTTA GATTACCAAT

3721 CCAAAAAGAA ACATGGGAGA CATGGTGGAC AGACTATTGG CAAGCCACCT GGATTCCTGA

3781 GTGGGAATTT GTTAATACCC CTCCCTTAGT AAAATTATGG TACCAACTGG AGAAAGATCC

3841 CATAGCAGGA GTAGAAACTT TCTATGTAGA TGGAGCAGCT AATAGGGAGA CTAAAATAGG

3901 GAAAGCAGGG TATGTTACTG ACAGAGGAAG GAAGAAAATT GTTTCCCTAA CTGAAACAAC

3961 AAACCAGAAG ACTGAATTGC AAGCAATTCA TATAGCTTTG AAAGATTCAG GATCAGAAGT

4021 AAACATAGTA ACAGATTCAC AGTATGCATT AGGGATCATT CAAGCACAAC CAGATAAAAG

4081 TGAATCAGAG TTAGTCAACC AAATAATAGA ACAATTAATA AAAAAGGAAA GGGTCTACCT

4141 GTCATGGGTA CCAGCACATA AAGGCATTGG AGGAAACGAA CAAGTAGATA AATTAGTAAG

4201 TAGTGGAATC AGGAAAGTGC TATTTTTAGA TGGAATAGAT AAAGCTCAAG AAGAGCATGA

4261 AAAGTACCAC AGCAATTGGA GAGCAATGGC TAGTGACTTT AATCTGCCAC CCATAGTAGC

4321 AAAAGAAATA GTAGCTAGCT GTGATAAATG TCAGCTAAAA GGGGAAGCCA TGCATGGACA

4381 AGTAGACTGT AGTCCAGGGA TATGGCAATT AGATTGTACA CATTTAGAAG GAAAAATCAT

4441 TCTGGTAGCA GTCCATGTAG CCAGTGGCTA CATGGAAGCA GAGGTTATCC CAGCAGAAAC

4501 AGGACAAGAA ACAGCATACT TTATACTAAA ATTAGCAGGA AGATGGCCAG TCAAAGTAAT

4561 ACATACAGAC AATGGTAGTA ATTTCACCAG TACTGCAGTT AAGGCAGCCT GTTGGTGGGC

4621 AGGTATCCGA CAGGAATTCG GAATTCCCTA CAATCCCCAA AGTCAGGGAG TAGTAGAATC

4681 CATGAATAAA GAATTAAAGA AACTTATAGG GCAGGTAAGA GATCAAGCTG AGCACCTTAA

4741 GACAGCAGTA CAAATGGCAG TATTCATTCA CAATTTTAAA AGAAAAGGGG GGATTGGGGG

4801 GTACAGTGCA GGGGAAAGAA TAGTAGACAT AATAGCAACA GACATACAAA CTAGAGAATT

4861 ACAAAAACAA ATTATAAAAA TTCAAAATTT TCGGGTTTAT TACAGAGACA GCAGAGACCC

4921 CATTTGGAAA GGACCAGCCA AACTACTCTG GAAAGGTGAA GGGGCAGTAG TAATACAAGA

4981 TAATAGTGAC ATAAAGGTAG TACCAAGGAG GAAAGCAAAA ATCATTAAGG ACTATGGAAA

5041 ACAGATGGCA GGTGCTGATT GTGTGGCAGG TAGACAGGAT GAAGATTAGA ACATGGAATA

5101 GTTTAGTAAA ACACCATATG TATATTTCAA GGAGAGCTAA GGGATGGTTT TACAGACATC

5161 ATTATGACAG CAGACATCCA AAGGTAAGTT CAGAAGTACA CATCCCATTG GGAGAGGCTA

5221 GATTAGTAAT AAAAACATAT TGGGGGTTGC AAACAGGAGA AAGAGATTGG CATTTGGGTC

5281 ATGGAGTCTC CATAGAATGG AGATTGAGAA GCTATGACAC ACAAATAGAA CCTGGCCTGG

5341 CAGACCAGCT AATTCATATG TATTATTTTG ATTGTTTTGC AGACTCTGCC ATAAGGAAAG

5401 CCATATTAGG ACACGTAGTT ATTCCTAGGT GTGACTATCA AGCAGGACAT AATAATAAGG

5461 TAGGATCTCT ACAATACTTG GCTCTGACAG CACTGATAAA ACCAAAAAAG ATAAAGCCAC

5521 CTCTGCCTAG TATTAAGAAA TTAGTAGAGG ATAGATGGAA CAACCCCCAG AAGATCACGG

5581 GCCGCAGAGG GAACCATACA ATGAATGGAC ACTAGAGCCT CTAGAGGAAC TCAAGCAGGA

5641 AGCTGTCAGA CACTTTCCTA GACCATGGCT TCATGGCTTA GGACAATATG TCTATGAAAA

5701 CTATGGGGAT ACTTGGACAG GGGTTGAAAC TTTAATAAGA ATACTGCAAC AATTACTGTT

5761 TATTCATTTC AGAATTGGGT GCCAGCATAG CAGAATAGGC ATTGTGAGAC AGAGAAGAGC

5821 AAGAAATGGA GCCAGTAGAT CCTAACCTAG AGCCCTGGAA CCATCCAGGA AGTCAGCCTA

5881 AAACTGCTTG TAATAATTGC TATTGTAAAC GCTGTAGCTA CCATTGTCCA GTTTGCTTTC

5941 TGACAAAAGG CTTAGGCATT TCCTATGGCA GGAAGAAGCG GAGACAGCGA CGAAGAGCTT

6001 CTCAGAGCAG TGAGGATCAT CAAAATCTTA TATCAAAGCA GTAAGTATCT GTAATGTTAA

6061 ATTTAGAATT AGCAATAGCA GTAGGAGCAT TGATAGTAGC ACTAATCATA GCAATAGTTG

6121 TGTGGACCAT AGTATATATA GAATATAGGA GATTGGTAAA ACAAAGAAAA ATAGGTTGGT

6181 TAATTAAAAG AATTAGGGAA AGAGCAGAAG ACAGTGGCAA TGAGAGTGAA GGGGACACTG

6241 AGGAATTATC AACAATGGTG GATATGGGGC GTCTTAGGCT TTTGGATGTT AATGATTTGT

6301 AATGTGGGAG GAAACTTGTG GGTCACAGTC TATTATGGGG TACCTGTGTG GAAAGATGCA

6361 AAAACTACCC TATTCTGTGC GTCAGATGCT AAAGCACATG AGACAGAAGT GCATAATGTC

6421 TGGGCTACAC ATGCCTGTGT ACCCACAGAC CCCAACCCAC AAGAAATAGT TATGGAAAAT

6481 GTAACAGAAA ATTTTAACAT GTGGAAAAAT GATATGGTGG ATCAGATGCA TGAGGATGTA

6541 ATCAGTTTAT GGGATCAAAG CCTAAAGCCA TGTGTAAAGT TGACCCCACT CTGTGTCACT

6601 TTAGAATGTA AAAATGTTAA TGGTACCTAC AATAGGACCT CCGATGAGAG CGTAAAGGAG

6661 ATAAAAAATT GCTCTTTCAA TGCAACCACA TTACTAACAG ATAAGAAGAA GAAAGTGTAT

6721 GCACTTTTTT ATAGACTTGA TATAGTACCA CTTAATGAGA AGAACTCCAG TAACTCTAAT

6781 GAGTCTTATA GATTAATAAA TTGTAATACC TCAGCCGTAA CACAAGCCTG TCCAAAGGTC

6841 ACTTTTGATC CAATTCCTAT ACACTATTGC ACTCCAGCTG GTTATGCGAT TCTACAGTGT

6901 AATAATAAGG CATTCAATGG GACAGGACCA TGCCATAATG TTAGCACGGT ACAATGTACA

6961 CATGGAATTA AGCCAGTGGT ATCAACTCAA CTACTGTTAA ATGGTAGCCT AGCAGAAAGA

7021 GAGATAATAA TTAGATCTGA AAATCTGACA AACAATGCCA AAACAATAAT AGTACATCTT

7081 AATGAATCTG TAGAAATTGT ATGTACAAGA CCCAACAATA ATACAAGAAA AAGTATAAGG

7141 ATAGGACCAG GACAAACATT CTATGCAACA GGAGAAATCA TAGGAGACAT AAGACAAGCA

7201 CATTGTAACA TTAGTGAAGA TAGTTGGAAT AAAACTTTAT GGAAGGTAAG TAAAAAATTA

7261 GCAGAATACT TCCCTAATAA AACAATAAAC TTTACATCAT CCTCAGGAGG GGACCTAGAA

7321 ATTACAACAC ATAGCTTTAA TTGTAGAGGA GAATTTTTCT ATTGTAATAC ATCAAAACTG

7381 TTTAACAGTA CATACAATAG TACAACAGGC CTGTTTAACG GTACATACAA TAGTACAAAA

7441 GAAGGTAATT CAAGCTCAAC CATCATACTT CCATGCAGAA TAAAGCAAAT TATAAACATG

7501 TGGCAGGAGG TAGGACGAGC AATGTATGCC CCTCCTATTG AAGGAAACAT AACATGTAAA

7561 TCAACTATCA CAGGACTATT ATTGGTACGT GATGGAGGAA AGACAAATGG TACAGAGGCA

7621 AATAGAACAG AGATATTCAG ACCTGGAGGA GGAGATATGA GGGACAATTG GAGAAGTGAA

7681 TTATATAAAT ATAAAGTGGT AGAAATTAAG CCATTGGGAG TAGCACCCAC TGCAGCAAAA

7741 AGGAGAGTGG TGGAGAGAGA AAAAAGAGCA GTGGGACTGG GAGCTGTGTT CCTTGGGTTC

7801 TTGGGAGCAG CAGGAAGCAC TATGGGCGCG GCGTCAATAA CGCTGACGGT ACAAGCCAGA

7861 CAATTGTTGT CTGGTATAGT GCAACAGCAA AGCAATTTGC TGAGAGCTAT AGAGGCGCAA

7921 CAGCATCTGT TGCAACTCAC GGTTTGGGGC ATTAAACAGC TACAGACAAG AGTCCTGGCT

7981 ATAGAAAGAT ACCTAAAGGA TCAACAGCTC CTAGGGATTT GGGGCTGCTC TGGAAAACTC

8041 ATCTGCACTA CTGCTGTACC TTGGAACTCC AGTTGGAGTA ACAAGTCTTA CACAGAGATT

8101 TGGGATAACA TGACCTGGAT GCAGTGGGAT AAGGAAATTA GTAATTACAC AAACACAATC

8161 TACAGGTTGC TTGAAGACTC GCAAAACCAG CAGGAAAGAA ATGAAAAAGA TTTATTGGCA

8221 TTGGACAGTT GGAAAAATCT ATGGAGTTGG TTTGACATAA CAAATTGGCT GTGGTATATA

8281 AGAATATTCA TAATGATAAT AGGAGGCTTG ATAGGTTTAA GAATAATTTT TGCTGTGCTT

8341 TCTATAGTGA ATAGAGTTAG GCAGGGATAC TCACCTTTGC CGTTTCAGAC CCTTACCCCG

8401 AACCCAGGGG GACCAGACAG GCTCAGAGGA ATCGAAGAAG AAGGTGGAGA GCAAGACAAA

8461 GCCAGATCCA TTCGATTAGT GAACGGATTC TTAGCACTTG CCTGGGACGA CCTGCGGAAC

8521 CTGTGCCTCT TCAGTTACCA CCGCTTGAGA GACTTCATAT TACTGACAGC GAGAGGAGTG

8581 GAACTTCTGG GACGCAACAG CCTCACGGGA CTACAGAGGG GGTGGGAAGC CCTTAAATAC

8641 CTGGGAAGTC TTGTGCAGTA TTGGGGTCTG GAACTAAAAA AGAGTACTAT TAGTCTGTTT

8701 GATACCATAG CAATAGCAGT AGCTGAAGGA ACAGACAGGA TTATAAACAT AGTACAAGGA

8761 ATTTGTAGAG CTATCCGCAA CATACCTAGA AGAATAAGAC AGGGCTTTGA AGCAGCTTTG

8821 CAATAAAATG GGGGGCAAGT GGTCAAAAAG TAGCATAGTT GGATGGCCTG CTATAAGAGA

8881 AAGAATAAGG CGAACTGAGC CAGCAGCAGA TGGGGTGGGA GCAGTATCTC GAGACCTGGA

8941 AAACCGTGGA GCAATCACAA GTAGCAACAC AGCAGCTACT AATGCTGATT GTGCCTGGCT

9001 AGAAGCACAA GAGGAGGAGG AGGTGGGTTT TCCAGTCAGA CCTCAGGTAC CTTTAAGGCC

9061 AATGACTTTT AAGGGAGCAT TTGATCTCAG CTTCTTTTTA AGAGAAAAGG GGGGACTGGA

9121 AGGGTTAATT TACTCTAAGA AAAGGCAAGA GATCCTTGAC TTGTGGGTCT ATCACACACA

9181 AGGCTACTTC CCTGATTGGC AAAACTACAC GCCGGGACCA GGGGTCAGAT TCCCACTGAC

9241 TTTTGGGTGG TGCTCCAAGC TAGTACCAGT TGACCCAAGG GAAGTAGAAG AGGCCAACGA

9301 AGGAGAAGAC AACTGCTTGC TACACCCTGT CTGCCAGCAT GGAATGGATG ATGAACACGG

9361 AGAAGTATTA AAGTGGAAGT TTGACAGTCA ACTAGCAAGC AGACACATGG CCCGCGAGCT

9421 ACATCCGGAG TTTTACAAAG ACTGCTGACA CAGAAGGGAC TGTCTGCTGA CACAGAAGGG

9481 ACTTTCCGCG GGACTTTCCA CTGGGGCGTT CCGGGAGGTG TGGTCTGGGC GGGACTGGGA

9541 GTGGTCAACC CTCAGATGCT GCATATAAGC AGCTGCTTTT CGCCTGTACT GGGTCTCTCT

9601 AGTTAGACCA GATCTGAGCC TGGGAGCTCT CTGGCTAGCT AGGGAACCCA CTGCTTAAGC

9661 CTCAATAAAG CTTGCCTTGA GTGCTCTGAG CAGTGTGTGC CCGTCTATTG TGTGACTCTG

9721 GTAACTAGAG ATCCCTCAGA CC

<210> 18

<211> 13384

<212> DNA

＜213〉artificial sequence (SHIV-KB9/pBR322 infections clone whole genome sequence)

<220>

<221>

<222>

<223>

<400> 18

1 GAATTCTAGA CATATACTTA GAAAAGGAAG AAGGCATCAT ACCAGATTGG CAGGATTATA

61 CCTCAGGACC AGGAATTAGA TACCCAAAGA CATTTGGCTG GCTATGGAAA TTAGTCCCTG

121 TAAATGTATC AGATGAGGCA CAGGAGGATG AGGAGCATTA TTTAATGCAT CCAGCTCAAA

181 CTTCCCAGTG GGATGACCCT TGGGGAGAGG TTCTAGCATG GAAGTTTGAT CCAACTCTGG

241 CCTACACTTA TGAGGCATAT GTTAGATACC CAGAAGAGTT TGGAAGCAAG TCAGGCCTGT

301 CAGAGGAAGA GGTTAGAAGA AGGCTAACCG CAAGAGGCCT TCTTAACATG GCTGACAAGA

361 AGGAAACTCG CTGAAACAGC AGGGACTTTC CACAAGGGGA TGTTACGGGG AGGTACTGGG

421 GAGGAGCCGG TCGGGAACGC CCACTTTCTT GATGTATAAA TATCACTGCA TTTCGCTCTG

481 TATTCAGTCG CTCTGCGGAG AGGCTGGCAG ATTGAGCCCT GGGAGGTTCT CTCCAGCACT

541 AGCAGGTAGA GCCTGGGTGT TCCCTGCTAG ACTCTCACCA GCACTTGGCC GGTGCTGGGC

601 AGAGTGACTC CACGCTTGTT TGCTTAAAGC CCTCTTCAAT AAAGCTGCCA TTTTAGAAGT

661 AAGCTAGTGT GTGTTCCCAT CTCTCCTAGC CGCCGCCTGG TCAACTCGGT ACTCAATAAT

721 AAGAAGACCC TGGTCTGTTA GGACCCTTTC TGCTTTGGGA AACCGAAGCA GGAAAATCCC

781 TAGCAGATTG GCGCCTGAAC AGGGACTTGA AGGAGAGTGA GAGACTCCTG AGTACGGCTG

841 AGTGAAGGCA GTAAGGGCGG CAGGAACCAA CCACGACGGA GTGCTCCTAT AAAGGCGCGG

901 GTCGGTACCA GACGGCGTGA GGAGCGGGAG AGGAAGAGGC CTCCGGTTGC AGGTAAGTGC

961 AACACAAAAA AGAAATAGCT GTCTTTTATC CAGGAAGGGG TAATAAGATA GAGTGGGAGA

1021 TGGGCGTGAG AAACTCCGTC TTGTCAGGGA AGAAAGCAGA TGAATTAGAA AAAATTAGGC

1081 TACGACCCAA CGGAAAGAAA AAGTACATGT TGAAGCATGT AGTATGGGCA GCAAATGAAT

1141 TAGATAGATT TGGATTAGCA GAAAGCCTGT TGGAGAACAA AGAAGGATGT CAAAAAATAC

1201 TTTCGGTCTT AGCTCCATTA GTGCCAACAG GCTCAGAAAA TTTAAAAAGC CTTTATAATA

1261 CTGTCTGCGT CATCTGGTGC ATTCACGCAG AAGAGAAAGT GAAACACACT GAGGAAGCAA

1321 AACAGATAGT GCAGAGACAC CTAGTGGTGG AAACAGGAAC AACAGAAACT ATGCCAAAAA

1381 CAAGTAGACC AACAGCACCA TCTAGCGGCA GAGGAGGAAA TTACCCAGTA CAACAAATAG

1441 GTGGTAACTA TGTCCACCTG CCATTAAGCC CGAGAACATT AAATGCCTGG GTAAAATTGA

1501 TAGAGGAAAA GAAATTTGGA GCAGAAGTAG TGCCAGGATT TCAGGCACTG TCAGAAGGTT

1561 GCACCCCCTA TGACATTAAT CAGATGTTAA ATTGTGTGGG AGACCATCAA GCGGCTATGC

1621 AGATTATCAG AGATATTATA AACGAGGAGG CTGCAGATTG GGACTTGCAG CACCCACAAC

1681 CAGCTCCACA ACAAGGACAA CTTAGGGAGC CGTCAGGATC AGATATTGCA GGAACAACTA

1741 GTTCAGTAGA TGAACAAATC CAGTGGATGT ACAGACAACA GAACCCCATA CCAGTAGGCA

1801 ACATTTACAG GAGATGGATC CAACTGGGGT TGCAAAAATG TGTCAGAATG TATAACCCAA

1861 CAAACATTCT AGATGTAAAA CAAGGGCCAA AAGAGCCATT TCAGAGCTAT GTAGACAGGT

1921 TCTACAAAAG TTTAAGAGCA GAACAGACAG ATGCAGCAGT AAAGAATTGG ATGACTCAAA

1981 CACTGCTGAT TCAAAATGCT AACCCAGATT GCAAGCTAGT GCTGAAGGGG CTGGGTGTGA

2041 ATCCCACCCT AGAAGAAATG CTGACGGCTT GTCAAGGAGT AGGGGGGCCG GGACAGAAGG

2101 CTAGATTAAT GGCAGAAGCC CTGAAAGAGG CCCTCGCACC AGTGCCAATC CCTTTTGCAG

2161 CAGCCCAACA GAGGGGACCA AGAAAGCCAA TTAAGTGTTG GAATTGTGGG AAAGAGGGAC

2221 ACTCTGCAAG GCAATGCAGA GCCCCAAGAA GACAGGGATG CTGGAAATGT GGAAAAATGG

2281 ACCATGTTAT GGCCAAATGC CCAGACAGAC AGGCGGGTTT TTTAGGCCTT GGTCCATGGG

2341 GAAAGAAGCC CCGCAATTTC CCCATGGCTC AAGTGCATCA GGGGCTGATG CCAACTGCTC

2401 CCCCAGAGGA CCCAGCTGTG GATCTGCTAA AGAACTACAT GCAGTTGGGC AAGCAGCAGA

2461 GAGAAAAGCA GAGAGAAAGC AGAGAGAAGC CTTACAAGGA GGTGACAGAG GATTTGCTGC

2521 ACCTCAATTC TCTCTTTGGA GGAGACCAGT AGTCACTGCT CATATTGAAG GACAGCCTGT

2581 AGAAGTATTA CTGGATACAG GGGCTGATGA TTCTATTGTA ACAGGAATAG AGTTAGGTCC

2641 ACATTATACC CCAAAAATAG TAGGAGGAAT AGGAGGTTTT ATTAATACTA AAGAATACAA

2701 AAATGTAGAA ATAGAAGTTT TAGGCAAAAG GATTAAAGGG ACAATCATGA CAGGGGACAC

2761 CCCGATTAAC ATTTTTGGTA GAAATTTGCT AACAGCTCTG GGGATGTCTC TAAATTTTCC

2821 CATAGCTAAA GTAGAGCCTG TAAAAGTCGC CTTAAAGCCA GGAAAGGATG GACCAAAATT

2881 GAAGCAGTGG CCATTATCAA AAGAAAAGAT AGTTGCATTA AGAGAAATCT GTGAAAAGAT

2941 GGAAAAGGAT GGTCAGTTGG AGGAAGCTCC CCCGACCAAT CCATACAACA CCCCCACATT

3001 TGCTATAAAG AAAAAGGATA AGAACAAATG GAGAATGCTG ATAGATTTTA GGGAACTAAA

3061 TAGGGTCACT CAGGACTTTA CGGAAGTCCA ATTAGGAATA CCACACCCTG CAGGACTAGC

3121 AAAAAGGAAA AGAATTACAG TACTGGATAT AGGTGATGCA TATTTCTCCA TACCTCTAGA

3181 TGAAGAATTT AGGCAGTACA CTGCCTTTAC TTTACCATCA GTAAATAATG CAGAGCCAGG

3241 AAAACGATAC ATTTATAAGG TTCTGCCTCA GGGATGGAAG GGGTCACCAG CCATCTTCCA

3301 ATACACTATG AGACATGTGC TAGAACCCTT CAGGAAGGCA AATCCAGATG TGACCTTAGT

3361 CCAGTATATG GATGACATCT TAATAGCTAG TGACAGGACA GACCTGGAAC ATGACAGGGT

3421 AGTTTTACAG TCAAAGGAAC TCTTGAATAG CATAGGGTTT TCTACCCCAG AAGAGAAATT

3481 CCAAAAAGAT CCCCCATTTC AATGGATGGG GTACGAATTG TGGCCAACAA AATGGAAGTT

3541 GCAAAAGATA GAGTTGCCAC AAAGAGAGAC CTGGACAGTG AATGATATAC AGAAGTTAGT

3601 AGGAGTATTA AATTGGGCAG CTCAAATTTA TCCAGGTATA AAAACCAAAC ATCTCTGTAG

3661 GTTAATTAGA GGAAAAATGA CTCTAACAGA GGAAGTTCAG TGGACTGAGA TGGCAGAAGC

3721 AGAATATGAG GAAAATAAAA TAATTCTCAG TCAGGAACAA GAAGGATGTT ATTACCAAGA

3781 AGGCAAGCCA TTAGAAGCCA CGGTAATAAA GAGTCAGGAC AATCAGTGGT CTTATAAAAT

3841 TCACCAAGAA GACAAAATAC TGAAAGTAGG AAAATTTGCA AAGATAAAGA ATACACATAC

3901 CAATGGAGTG AGACTATTAG CACATGTAAT ACAGAAAATA GGAAAGGAAG CAATAGTGAT

3961 CTGGGGACAG GTCCCAAAAT TCCACTTACC AGTTGAGAAG GATGTATGGG AACAGTGGTG

4021 GACAGACTAT TGGCAGGTAA CCTGGATACC GGAATGGGAT TTTATCTCAA CACCACCGCT

4081 AGTAAGATTA GTCTTCAATC TAGTGAAGGA CCCTATAGAG GGAGAAGAAA CCTATTATAC

4141 AGATGGATCA TGTAATAAAC AGTCAAAAGA AGGGAAAGCA GGATATATCA CAGATAGGGG

4201 CAAAGACAAA GTAAAAGTGT TAGAACAGAC TACTAATCAA CAAGCAGAAT TGGAAGCATT

4261 TCTCATGGCA TTGACAGACT CAGGGCCAAA GGCAAATATT ATAGTAGATT CACAATATGT

4321 TATGGGAATA ATAACAGGAT GCCCTACAGA ATCAGAGAGC AGGCTAGTTA ATCAAATAAT

4381 AGAAGAAATG ATTAAAAAGT CAGAAATTTA TGTAGCATGG GTACCAGCAC ACAAAGGTAT

4441 AGGAGGAAAC CAAGAAATAG ACCACCTAGT TAGTCAAGGG ATTAGACAAG TTCTCTTCTT

4501 GGAAAAGATA GAGCCAGCAC AAGAAGAACA TGATAAATAC CATAGTAATG TAAAAGAATT

4561 GGTATTCAAA TTTGGATTAC CCAGAATAGT GGCCAGACAG ATAGTAGACA CCTGTGATAA

4621 ATGTCATCAG AAAGGAGAGG CTATACATGG GCAGGCAAAT TCAGATCTAG GGACTTGGCA

4681 AATGGATTGT ACCCATCTAG AGGGAAAAAT AATCATAGTT GCAGTACATG TAGCTAGTGG

4741 ATTCATAGAA GCAGAGGTAA TTCCACAAGA GACAGGAAGA CAGACAGCAC TATTTCTGTT

4801 AAAATTGGCA GGCAGATGGC CTATTACACA TCTACACACA GATAATGGTG CTAACTTTGC

4861 TTCGCAAGAA GTAAAGATGG TTGCATGGTG GGCAGGGATA GAGCACACCT TTGGGGTACC

4921 ATACAATCCA CAGAGTCAGG GAGTAGTGGA AGCAATGAAT CACCACCTGA AAAATCAAAT

4981 AGATAGAATC AGGGAACAAG CAAATTCAGT AGAAACCATA GTATTAATGG CAGTTCATTG

5041 CATGAATTTT AAAAGAAGGG GAGGAATAGG GGATATGACT CCAGCAGAAA GATTAATTAA

5101 CATGATCACT ACAGAACAAG AGATACAATT TCAACAATCA AAAAACTCAA AATTTAAAAA

5161 TTTTCGGGTC TATTACAGAG AAGGCAGAGA TCAACTGTGG AAGGGACCCG GTGAGCTATT

5221 GTGGAAAGGG GAAGGAGCAG TCATCTTAAA GGTAGGGACA GACATTAAGG TAGTACCCAG

5281 AAGAAAGGCT AAAATTATCA AAGATTATGG AGGAGGAAAA GAGGTGGATA GCAGTTCCCA

5341 CATGGAGGAT ACCGGAGAGG CTAGAGAGGT GGCATAGCCT CATAAAATAT CTGAAATATA

5401 AAACTAAAGA TCTACAAAAG GTTTGCTATG TGCCCCATTT TAAGGTCGGA TGGGCATGGT

5461 GGACCTGCAG CAGAGTAATC TTCCCACTAC AGGAAGGAAG CCATTTAGAA GTACAAGGGT

5521 ATTGGCATTT GACACCAGAA AAAGGGTGGC TCAGTACTTA TGCAGTGAGG ATAACCTGGT

5581 ACTCAAAGAA CTTTTGGACA GATGTAACAC CAAACTATGC AGACATTTTA CTGCATAGCA

5641 CTTATTTCCC TTGCTTTACA GCGGGAGAAG TGAGAAGGGC CATCAGGGGA GAACAACTGC

5701 TGTCTTGCTG CAGGTTCCCG AGAGCTCATA AGTACCAGGT ACCAAGCCTA CAGTACTTAG

5761 CACTGAAAGT AGTAAGCGAT GTCAGATCCC AGGGAGAGAA TCCCACCTGG AAACAGTGGA

5821 GAAGAGACAA TAGGAGAGGC CTTCGAATGG CTAAACAGAA CAGTAGAGGA GATAAACAGA

5881 GAGGCGGTAA ACCACCTACC AAGGGAGCTA ATTTTCCAGG TTTGGCAAAG GTCTTGGGAA

5941 TACTGGCATG ATGAACAAGG GATGTCACCA AGCTATGTAA AATACAGATA CTTGTGTTTA

6001 ATACAAAAGG CTTTATTTAT GCATTGCAAG AAAGGCTGTA GATGTCTAGG GGAAGGACAT

6061 GGGGCAGGGG GATGGAGACC AGGACCTCCT CCTCCTCCCC CTCCAGGACT AGCATAAATG

6121 GAAGAAAGAC CTCCAGAAAA TGAAGGACCA CAAAGGGAAC CATGGGATGA ATGGGTAGTG

6181 GAGGTTCTGG AAGAACTGAA AGAAGAAGCT TTAAAACATT TTGATCCTCG CTTGCTAACT

6241 GCACTTGGTA ATCATATCTA TAATCGTCAC GGAGACACTC TAGAGGGAGC AGGAGAACTC

6301 ATTAGAATCC TCCAACGAGC GCTCTTCATG CATTTCAGAG GCGGATGCAT CCACTCCAGA

6361 ATCGGCCAAC CTGGGGGAGG AAATCCTCTC TCAGCTATAC CGCCCTCTAG AAGCATGCTG

6421 TAGAGCAAGA AATGGAGCCA GTAGATCCTA GACTAGAGCC CTGGAAGCAT CCAGGGAGTA

6481 AGCCTAAAAC TGCTTGTACC AATTGCTATT GTAAAAAGTG TTGCTTTCAT TGCCAAGTTT

6541 GTTTCACAAC AAAAGCCTTA GGCATCTCCT ATGGCAGGAA GAAGCGGAGA CAGCGACGAA

6601 GAGCTCATCA GAACAGTCAG ACTCATCAAG CTTCTCTATC AAAGCAGTAA GTAGTACATG

6661 TAATGCAACC TATACAAATA GCAATAGTAG CATTAGTAGT AGCAATAATA ATAGCAATAG

6721 TTGTGTGGTC CATAGTAATC ATAGAATATA GGAAAATATT AAGACAAAGA AAAATAGACA

6781 GGTTAATTGA TAGACTAATA GAAAGAGCAG AAGACAGTGG CAATGAGAGT GAAGGAGAAA

6841 TATCAGCACT TGTGGAGATG GGGGTGGAGA TGGGGCACCA TGCTCCTTGG GATGTTGATG

6901 ATCTGTAGTG CTACAGAAAA ATTGTGGGTC ACAGTCTATT ATGGGGTACC TGTGTGGAGA

6961 GAAGCAACCA CCACTCTATT TTGTGCATCA GATGCTAAAG CCTATGATAC AGAGGTACAT

7021 AATGTTTGGG CCACACATGC CTGTGTACCC ACAGACCCCA ACCCACAAGA AGTAGTATTG

7081 GGAAATGTGA CAGAAAATTT TAACATGTGG AAAAATAACA TGGTAGATCA GATGCATGAG

7141 GATATAATCA GTTTATGGGA TGAAAGCCTA AAGCCATGTG TAAAATTAAC CCCACTCTGT

7201 GTTACTTTAA ATTGCACTAA TTTGAATATC ACTAAGAATA CTACTAATCT CACTAGTAGC

7261 AGCTGGGGAA TGATGGAGGA AGGAGAAATA AAAAATTGCT CTTTCTATAT CACCACAAGC

7321 ATAAGAAATA AGGTAAAGAA AGAATATGCA CTTTTTAATA GACTTGATGT AGTACCAGTA

7381 AAAAATACTA GTAATACTAA GTATAGGTTA ATAAGTTGTA ACACCTCAGT CATTACACAG

7441 GCCTGTCCAA AGGTATCCTT TCAGCCAATT CCCATACATT ATTGTGTCCC GGCTGGGTTT

7501 GCGATACTAA AGTGTAACAA TAAGACATTC AATGGATCAG GACCATGCAC AAATGTCAGC

561 ACAGTACAAT GTACACATGG AATTAGGCCA GTGGTGTCAA CTCAACTGCT GTTAAATGGC

7621 AGTCTAGCAG AAGAAGACAT AGTAATTAGA TCTGAAGATT TCACAGACAA TGTTAAAACC

7681 ATAATAGTAC AGCTAAATGA ATCTGTAGTA ATTAATTGTA CAAGACCCAA CAACAATACA

7741 AGAGAAAGGT TATCTATAGG ACCAGGGAGA GCATTTTATG CAAGAAGAAA CATAATAGGA

7801 GATATAAGAC AAGCACATTG TAACATTAGT AGAGCAAAAT GGAATAACAC TTTACAACAG

7861 ATAGTTATAA AATTAAGAGA AAAATTTAGG AATAAAACAA TAGCCTTTAA TCAATCCTCA

7921 GGAGGGGACC CAGAAATTGT AATGCACAGT TTTAATTGTG GAGGGGAATT TTTCTACTGT

7981 AATACAGCAC AACTGTTTAA TAGTACTTGG AATGTTGCTG GAGGGACAAA TGGCACTGAA

8041 GGAAATGACA TAATCACACT CCAATGCAGA ATAAAACAAA TTATAAATAT GTGGCAGAAA

8101 GTAGGAAAAG CAATGTATGC CCCTCCCATC ACAGGACAAA TTAGATGTTC ATCAAATATT

8161 ACAGGGCTGC TACTAACAAG AGATGGAGGT AATAGTACTG AGACTGAGAC TGAGATCTTC

8221 AGACCTGGAG GAGGAGATAT GAGGGACAAT TGGAGAAGTG AATTATATAA ATATAAAGTA

8281 GTAAGAATTG AACCAATAGG AGTAGCACCC ACCAGGGCAA AGAGAAGAAC AGTGCAAAGA

8341 GAAAAAAGAG CAGTGGGAAT AGGAGCTGTG TTCCTTGGGT TCTTGGGAGC AGCAGGAAGC

8401 ACTATGGGCG CAGCGTCAGT GACGCTGACG GTACAGGCCA GGCTATTATT GTCTGGTATA

8461 GTGCAGCAGC AGAACAATCT GCTGAGGGCT ATTGAGGCGC AACAGAATAT GTTGCGACTC

8521 ACAGTCTGGG GCATCAAGCA GCTCCAGGCA AGAGTCCTGG CTCTGGAAAG ATACCTAAGG

8581 GATCAACAGC TCATGGGAAT TTGGGGTTGC TCTGGAAAAC TCATTTGCAC CACTTCTGTG

8641 CCTTGGAATG TTAGTTGGAG TAATAAATCT GTGGATGATA TTTGGAATAA CATGACCTGG

8701 ATGGAGTGGG AAAGAGAAAT TGACAATTAC ACAGACTATA TATATGACTT ACTTGAAAAA

8761 TCGCAAACCC AACAAGAAAA GAATGAAAAA GAATTATTGG AATTGGATAA ATGGGCAAGT

8821 TTGTGGAATT GGTTTGACAT AACAAACTGG CTGTGGTATA TAAGATTATT CATAATGATA

8881 GTAGGAGGCT TGATAGGTTT AAGAATAGTT TTTGCTGTAC TTTCTATAGT AAATAGAGTT

8941 AGGCAGGGAT ATTCACCATT ATCGTTTCAG ACCCTCCTCC CAGCCTCGAG GGGACCCGAC

9001 AGGCCCGAAG GAACAGAAGA AGAAGGTGGA GAGAGAGACA GAGACAGATCCGGTCCATCA

9061 GTGAACGGAT CCTTGGCACT TATCTGGGAC GATCTGCGGA GCCTGTGCCT CTTCAGCTAC

9121 CACCGCTTGA GAGACTTACT CTTGATTGTA ACGAGGATTG TGGAACTTCT GGGACGCAGG

9181 GGGTGGGAAG CCCTCAAATA TTGGTGGAAT CTCCTACAGT ATTGGAGTCA GGAACTAAAG

9241 AATAGTGCTG TTAGCTTGCT ACAATATGGG TGGAGCTATT TCCATGAGGC GGTCCAGGCC

9301 GTCTGGAGAT CTGCGACAGA GACTCTTGCG GGCGCGTGGG GAGACTTATG GGAGACTCTT

9361 AGGAGAGGTG GAAGATGGAT ACTCGCAATC CCCAGGAGGA TTAGACAAGG GCTTGAGCTC

9421 ACTCTCTTGT GAGGGACAGA AATACAATCA GGGACAGTAT ATGAATACTC CATGGAGAAA

9481 CCCAGCTGAA GAGAGAGAAA AATTAGCATA CAGAAAACAA AATATGGATG ATATAGATGA

9541 GGAAGATGAT GACTTGGTAG GGGTATCAGT GAGGCCAAAA GTTCCCCTAA GAACAATGAG

9601 TTACAAATTG GCAATAGACA TGTCTCATTT TATAAAAGAA AAGGGGGGAC TGGAAGGGAT

9661 TTATTACAGT GCAAGAAGAC ATAGAATCTT AGACATATAC TTAGAAAAGG AAGAAGGCAT

9721 CATACCAGAT TGGCAGGATT ATACCTCAGG ACCAGGAATT AGATACCCAA AGACATTTGG

9781 CTGGCTATGG AAATTAGTCC CTGTAAATGT ATCAGATGAG GCACAGGAGG ATGAGGAGCA

9841 TTATTTAATG CATCCAGCTC AAACTTCCCA GTGGGATGAC CCTTGGGGAG AGGTTCTAGC

9901 ATGGAAGTTT GATCCAACTC TGGCCTACAC TTATGAGGCA TATGTTAGAT ACCCAGAAGA

9961 GTTTGGAAGC AAGTCAGGCC TGTCAGAGGA AGAGGTTAGA AGAAGGCTAA CCGCAAGAGG

10021 CCTTCTTAAC ATGGCTGACA AGAAGGAAAC TCGCTGAAAC AGCAGGGACT TTCCACAAGG

10081 GGATGTTACG GGGAGGTACT GGGGAGGAGC CGGTCGGGAA CGCCCACTTT CTTGATGTAT

10141 AAATATCACT GCATTTCGCT CTGTATTCAG TCGCTCTGCG GAGAGGCTGG CAGATTGAGC

10201 CCTGGGAGGT TCTCTCCAGC ACTAGCAGGT AGAGCCTGGG TGTTCCCTGC TAGACTCTCA

10261 CCAGCACTTG GCCGGTGCTG GGCAGAGTGA CTCCACGCTT GTTTGCTTAA AGCCCTCTTC

10321 AATAAAGCTG CCATTTTAGA AGTAAGCTAG TGTGTGTTCC CATCTCTCCT AGCCGCCGCC

10381 TGGTCAACTC GGTACGCGGC CGcgtgatac gcctattttt ataggttaat gtcatgataa

10441 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt

10501 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa

10561 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta

10621 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag

10681 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca

10741 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta

10801 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc

10861 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc

10921 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca

10981 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc

11041 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca

11101 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac

11161 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg

11221 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg

11281 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg

11341 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac

11401 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc

11461 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct

11521 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc

11581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc

11641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg

11701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa

11761 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc

11821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt

11881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa

11941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc

12001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcggacaggtatc

12061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct

12121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat

12181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc

12241 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg

12301 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc

12361 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc

12421 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg

12481 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg

12541 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta

12601 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc

12661 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat

12721 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct

12781 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt

12841 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac

12901 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact

12961 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt

13021 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa

13081 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa

13141 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg

13201 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt

13261 cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat

13321 gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt

13381 ttgc

<210> 19

<211> 13408

<212> DNA

＜213〉artificial sequence (SHIV-KBQJ-12 infections clone whole genome sequence)

<220>

<221>

<222>

<223>

<400> 19

1 GAATTCTAGA CATATACTTA GAAAAGGAAG AAGGCATCAT ACCAGATTGG CAGGATTATA

61 CCTCAGGACC AGGAATTAGA TACCCAAAGA CATTTGGCTG GCTATGGAAA TTAGTCCCTG

121 TAAATGTATC AGATGAGGCA CAGGAGGATG AGGAGCATTA TTTAATGCAT CCAGCTCAAA

181 CTTCCCAGTG GGATGACCCT TGGGGAGAGG TTCTAGCATG GAAGTTTGAT CCAACTCTGG

241 CCTACACTTA TGAGGCATAT GTTAGATACC CAGAAGAGTT TGGAAGCAAG TCAGGCCTGT

301 CAGAGGAAGA GGTTAGAAGA AGGCTAACCG CAAGAGGCCT TCTTAACATG GCTGACAAGA

361 AGGAAACTCG CTGAAACAGC AGGGACTTTC CACAAGGGGA TGTTACGGGG AGGTACTGGG

421 GAGGAGCCGG TCGGGAACGC CCACTTTCTT GATGTATAAA TATCACTGCA TTTCGCTCTG

481 TATTCAGTCG CTCTGCGGAG AGGCTGGCAG ATTGAGCCCT GGGAGGTTCT CTCCAGCACT

541 AGCAGGTAGA GCCTGGGTGT TCCCTGCTAG ACTCTCACCA GCACTTGGCC GGTGCTGGGC

601 AGAGTGACTC CACGCTTGTT TGCTTAAAGC CCTCTTCAAT AAAGCTGCCA TTTTAGAAGT

661 AAGCTAGTGT GTGTTCCCAT CTCTCCTAGC CGCCGCCTGG TCAACTCGGT ACTCAATAAT

721 AAGAAGACCC TGGTCTGTTA GGACCCTTTC TGCTTTGGGA AACCGAAGCA GGAAAATCCC

781 TAGCAGATTG GCGCCTGAAC AGGGACTTGA AGGAGAGTGA GAGACTCCTG AGTACGGCTG

841 AGTGAAGGCA GTAAGGGCGG CAGGAACCAA CCACGACGGA GTGCTCCTAT AAAGGCGCGG

901 GTCGGTACCA GACGGCGTGA GGAGCGGGAG AGGAAGAGGC CTCCGGTTGC AGGTAAGTGC

961 AACACAAAAA AGAAATAGCT GTCTTTTATC CAGGAAGGGG TAATAAGATA GAGTGGGAGA

1021 TGGGCGTGAG AAACTCCGTC TTGTCAGGGA AGAAAGCAGA TGAATTAGAA AAAATTAGGC

1081 TACGACCCAA CGGAAAGAAA AAGTACATGT TGAAGCATGT AGTATGGGCA GCAAATGAAT

1141 TAGATAGATT TGGATTAGCA GAAAGCCTGT TGGAGAACAA AGAAGGATGT CAAAAAATAC

1201 TTTCGGTCTT AGCTCCATTA GTGCCAACAG GCTCAGAAAA TTTAAAAAGC CTTTATAATA

1261 CTGTCTGCGT CATCTGGTGC ATTCACGCAG AAGAGAAAGT GAAACACACT GAGGAAGCAA

1321 AACAGATAGT GCAGAGACAC CTAGTGGTGG AAACAGGAAC AACAGAAACT ATGCCAAAAA

1381 CAAGTAGACC AACAGCACCA TCTAGCGGCA GAGGAGGAAA TTACCCAGTA CAACAAATAG

1441 GTGGTAACTA TGTCCACCTG CCATTAAGCC CGAGAACATT AAATGCCTGG GTAAAATTGA

1501 TAGAGGAAAA GAAATTTGGA GCAGAAGTAG TGCCAGGATT TCAGGCACTG TCAGAAGGTT

1561 GCACCCCCTA TGACATTAAT CAGATGTTAA ATTGTGTGGG AGACCATCAA GCGGCTATGC

1621 AGATTATCAG AGATATTATA AACGAGGAGG CTGCAGATTG GGACTTGCAG CACCCACAAC

1681 CAGCTCCACA ACAAGGACAA CTTAGGGAGC CGTCAGGATC AGATATTGCA GGAACAACTA

1741 GTTCAGTAGA TGAACAAATC CAGTGGATGT ACAGACAACA GAACCCCATA CCAGTAGGCA

1801 ACATTTACAG GAGATGGATC CAACTGGGGT TGCAAAAATG TGTCAGAATG TATAACCCAA

1861 CAAACATTCT AGATGTAAAA CAAGGGCCAA AAGAGCCATT TCAGAGCTAT GTAGACAGGT

1921 TCTACAAAAG TTTAAGAGCA GAACAGACAG ATGCAGCAGT AAAGAATTGG ATGACTCAAA

1981 CACTGCTGAT TCAAAATGCT AACCCAGATT GCAAGCTAGT GCTGAAGGGG CTGGGTGTGA

2041 ATCCCACCCT AGAAGAAATG CTGACGGCTT GTCAAGGAGT AGGGGGGCCG GGACAGAAGG

2101 CTAGATTAAT GGCAGAAGCC CTGAAAGAGG CCCTCGCACC AGTGCCAATC CCTTTTGCAG

2161 CAGCCCAACA GAGGGGACCA AGAAAGCCAA TTAAGTGTTG GAATTGTGGG AAAGAGGGAC

2221 ACTCTGCAAG GCAATGCAGA GCCCCAAGAA GACAGGGATG CTGGAAATGT GGAAAAATGG

2281 ACCATGTTAT GGCCAAATGC CCAGACAGAC AGGCGGGTTT TTTAGGCCTT GGTCCATGGG

2341 GAAAGAAGCC CCGCAATTTC CCCATGGCTC AAGTGCATCA GGGGCTGATG CCAACTGCTC

2401 CCCCAGAGGA CCCAGCTGTG GATCTGCTAA AGAACTACAT GCAGTTGGGC AAGCAGCAGA

2461 GAGAAAAGCA GAGAGAAAGC AGAGAGAAGC CTTACAAGGA GGTGACAGAG GATTTGCTGC

2521 ACCTCAATTC TCTCTTTGGA GGAGACCAGT AGTCACTGCT CATATTGAAG GACAGCCTGT

2581 AGAAGTATTA CTGGATACAG GGGCTGATGA TTCTATTGTA ACAGGAATAG AGTTAGGTCC

2641 ACATTATACC CCAAAAATAG TAGGAGGAAT AGGAGGTTTT ATTAATACTA AAGAATACAA

2701 AAATGTAGAA ATAGAAGTTT TAGGCAAAAG GATTAAAGGG ACAATCATGA CAGGGGACAC

2761 CCCGATTAAC ATTTTTGGTA GAAATTTGCT AACAGCTCTG GGGATGTCTC TAAATTTTCC

2821 CATAGCTAAA GTAGAGCCTG TAAAAGTCGC CTTAAAGCCA GGAAAGGATG GACCAAAATT

2881 GAAGCAGTGG CCATTATCAA AAGAAAAGAT AGTTGCATTA AGAGAAATCT GTGAAAAGAT

2941 GGAAAAGGAT GGTCAGTTGG AGGAAGCTCC CCCGACCAAT CCATACAACA CCCCCACATT

3001 TGCTATAAAG AAAAAGGATA AGAACAAATG GAGAATGCTG ATAGATTTTA GGGAACTAAA

3061 TAGGGTCACT CAGGACTTTA CGGAAGTCCA ATTAGGAATA CCACACCCTG CAGGACTAGC

3121 AAAAAGGAAA AGAATTACAG TACTGGATAT AGGTGATGCA TATTTCTCCA TACCTCTAGA

3181 TGAAGAATTT AGGCAGTACA CTGCCTTTAC TTTACCATCA GTAAATAATG CAGAGCCAGG

3241 AAAACGATAC ATTTATAAGG TTCTGCCTCA GGGATGGAAG GGGTCACCAG CCATCTTCCA

3301 ATACACTATG AGACATGTGC TAGAACCCTT CAGGAAGGCA AATCCAGATG TGACCTTAGT

3361 CCAGTATATG GATGACATCT TAATAGCTAG TGACAGGACA GACCTGGAAC ATGACAGGGT

3421 AGTTTTACAG TCAAAGGAAC TCTTGAATAG CATAGGGTTT TCTACCCCAG AAGAGAAATT

3481 CCAAAAAGAT CCCCCATTTC AATGGATGGG GTACGAATTG TGGCCAACAA AATGGAAGTT

3541 GCAAAAGATA GAGTTGCCAC AAAGAGAGAC CTGGACAGTG AATGATATAC AGAAGTTAGT

3601 AGGAGTATTA AATTGGGCAG CTCAAATTTA TCCAGGTATA AAAACCAAAC ATCTCTGTAG

3661 GTTAATTAGA GGAAAAATGA CTCTAACAGA GGAAGTTCAG TGGACTGAGA TGGCAGAAGC

3721 AGAATATGAG GAAAATAAAA TAATTCTCAG TCAGGAACAA GAAGGATGTT ATTACCAAGA

3781 AGGCAAGCCA TTAGAAGCCA CGGTAATAAA GAGTCAGGAC AATCAGTGGT CTTATAAAAT

3841 TCACCAAGAA GACAAAATAC TGAAAGTAGG AAAATTTGCA AAGATAAAGA ATACACATAC

3901 CAATGGAGTG AGACTATTAG CACATGTAAT ACAGAAAATA GGAAAGGAAG CAATAGTGAT

3961 CTGGGGACAG GTCCCAAAAT TCCACTTACC AGTTGAGAAG GATGTATGGG AACAGTGGTG

4021 GACAGACTAT TGGCAGGTAA CCTGGATACC GGAATGGGAT TTTATCTCAA CACCACCGCT

4081 AGTAAGATTA GTCTTCAATC TAGTGAAGGA CCCTATAGAG GGAGAAGAAA CCTATTATAC

4141 AGATGGATCA TGTAATAAAC AGTCAAAAGA AGGGAAAGCA GGATATATCA CAGATAGGGG

4201 CAAAGACAAA GTAAAAGTGT TAGAACAGAC TACTAATCAA CAAGCAGAAT TGGAAGCATT

4261 TCTCATGGCA TTGACAGACT CAGGGCCAAA GGCAAATATT ATAGTAGATT CACAATATGT

4321 TATGGGAATA ATAACAGGAT GCCCTACAGA ATCAGAGAGC AGGCTAGTTA ATCAAATAAT

4381 AGAAGAAATG ATTAAAAAGT CAGAAATTTA TGTAGCATGG GTACCAGCAC ACAAAGGTAT

4441 AGGAGGAAAC CAAGAAATAG ACCACCTAGT TAGTCAAGGG ATTAGACAAG TTCTCTTCTT

4501 GGAAAAGATA GAGCCAGCAC AAGAAGAACA TGATAAATAC CATAGTAATG TAAAAGAATT

4561 GGTATTCAAA TTTGGATTAC CCAGAATAGT GGCCAGACAG ATAGTAGACA CCTGTGATAA

4621 ATGTCATCAG AAAGGAGAGG CTATACATGG GCAGGCAAAT TCAGATCTAG GGACTTGGCA

4681 AATGGATTGT ACCCATCTAG AGGGAAAAAT AATCATAGTT GCAGTACATG TAGCTAGTGG

4741 ATTCATAGAA GCAGAGGTAA TTCCACAAGA GACAGGAAGA CAGACAGCAC TATTTCTGTT

4801 AAAATTGGCA GGCAGATGGC CTATTACACA TCTACACACA GATAATGGTG CTAACTTTGC

4861 TTCGCAAGAA GTAAAGATGG TTGCATGGTG GGCAGGGATA GAGCACACCT TTGGGGTACC

4921 ATACAATCCA CAGAGTCAGG GAGTAGTGGA AGCAATGAAT CACCACCTGA AAAATCAAAT

4981 AGATAGAATC AGGGAACAAG CAAATTCAGT AGAAACCATA GTATTAATGG CAGTTCATTG

5041 CATGAATTTT AAAAGAAGGG GAGGAATAGG GGATATGACT CCAGCAGAAA GATTAATTAA

5101 CATGATCACT ACAGAACAAG AGATACAATT TCAACAATCA AAAAACTCAA AATTTAAAAA

5161 TTTTCGGGTC TATTACAGAG AAGGCAGAGA TCAACTGTGG AAGGGACCCG GTGAGCTATT

5221 GTGGAAAGGG GAAGGAGCAG TCATCTTAAA GGTAGGGACA GACATTAAGG TAGTACCCAG

5281 AAGAAAGGCT AAAATTATCA AAGATTATGG AGGAGGAAAA GAGGTGGATA GCAGTTCCCA

5341 CATGGAGGAT ACCGGAGAGG CTAGAGAGGT GGCATAGCCT CATAAAATAT CTGAAATATA

5401 AAACTAAAGA TCTACAAAAG GTTTGCTATG TGCCCCATTT TAAGGTCGGA TGGGCATGGT

5461 GGACCTGCAG CAGAGTAATC TTCCCACTAC AGGAAGGAAG CCATTTAGAA GTACAAGGGT

5521 ATTGGCATTT GACACCAGAA AAAGGGTGGC TCAGTACTTA TGCAGTGAGG ATAACCTGGT

5581 ACTCAAAGAA CTTTTGGACA GATGTAACAC CAAACTATGC AGACATTTTA CTGCATAGCA

5641 CTTATTTCCC TTGCTTTACA GCGGGAGAAG TGAGAAGGGC CATCAGGGGA GAACAACTGC

5701 TGTCTTGCTG CAGGTTCCCG AGAGCTCATA AGTACCAGGT ACCAAGCCTA CAGTACTTAG

5761 CACTGAAAGT AGTAAGCGAT GTCAGATCCC AGGGAGAGAA TCCCACCTGG AAACAGTGGA

5821 GAAGAGACAA TAGGAGAGGC CTTCGAATGG CTAAACAGAA CAGTAGAGGA GATAAACAGA

5881 GAGGCGGTAA ACCACCTACC AAGGGAGCTA ATTTTCCAGG TTTGGCAAAG GTCTTGGGAA

5941 TACTGGCATG ATGAACAAGG GATGTCACCA AGCTATGTAA AATACAGATA CTTGTGTTTA

6001 ATACAAAAGG CTTTATTTAT GCATTGCAAG AAAGGCTGTA GATGTCTAGG GGAAGGACAT

6061 GGGGCAGGGG GATGGAGACC AGGACCTCCT CCTCCTCCCC CTCCAGGACT AGCATAAATG

6121 GAAGAAAGAC CTCCAGAAAA TGAAGGACCA CAAAGGGAAC CATGGGATGA ATGGGTAGTG

6181 GAGGTTCTGG AAGAACTGAA AGAAGAAGCT TTAAAACATT TTGATCCTCG CTTGCTAACT

6241 GCACTTGGTA ATCATATCTA TAATCGTCAC GGAGACACTC TAGAGGGAGC AGGAGAACTC

6301 ATTAGAATCC TCCAACGAGC GCTCTTCATG CATTTCAGAG GCGGATGCAT CCACTCCAGA

6361 ATCGGCCAAC CTGGGGGAGG AAATCCTCTC TCAGCTATAC CGCCCTCTAG AAGCATGCTG

6421 TAGAGCAAGA AATGGAGCCA GTAGATCCTA GACTAGAGCC CTGGAAGCAT CCAGGGAGTA

6481 AGCCTAAAAC TGCTTGTACC AATTGCTATT GTAAAAAGTG TTGCTTTCAT TGCCAAGTTT

6541 GTTTCACAAC AAAAGCCTTA GGCATCTCCT ATGGCAGGAA GAAGCGGAGA CAGCGACGAA

6601 GAGCTCATCA GAACAGTCAG ACTCATCAAG CTTCTCTATC AAAGCAGTAA GTAGTACATG

6661 TAATGCAACC TATACAAATA GCAATAGTAG CATTAGTAGT AGCAATAATA ATAGCAATAG

6721 TTGTGTGGTC CATAGTAATC ATAGAATATA GGAAAATATT AAGACAAAGA AAAATAGACA

6781 GGTTAATTGA TAGACTAATA GAAAGAGCAG AAGACAGTGG CAATGAGAGT GAAGGAGAAA

6841 TATCAGCACT TGTGGAGATG GGGGTGGAGA TGGGGCACCA TGCTCCTTGG GATGTTGATG

6901 ATCTGTAGTG CTACAGAAAA ATTGTGGGTC ACAGTCTATT ATGGGGTACC TGTGTGGAAA

6961 GATGCAAAAA CTACCCTATT CTGTGCGTCA GATGCTAAAG CACATGAGAC AGAAGTGCAT

7021 AATGTCTGGG CTACACATGC CTGTGTACCC ACAGACCCCA ACCCACAAGA AATAGTTATG

7081 GAAAATGTAA CAGAAAATTT TAACATGTGG AAAAATGATA TGGTGGATCA GATGCATGAG

7141 GATGTAATCA GTTTATGGGA TCAAAGCCTA AAGCCATGTG TAAAGTTGAC CCCACTCTGT

7201 GTCACTTTAG AATGTAAAAA TGTTAATGGT ACCTACAATA GGACCTCCGA TGAGAGCGTA

7261 AAGGAGATAA AAAATTGCTC TTTCAATGCA ACCACATTAC TAACAGATAA GAAGAAGAAA

7321 GTGTATGCAC TTTTTTATAG ACTTGATATA GTACCACTTA ATGAGAAGAA CTCCAGTAAC

7381 TCTAATGAGT CTTATAGATT AATAAATTGT AATACCTCAG CCGTAACACA AGCCTGTCCA

7441 AAGGTCACTT TTGATCCAAT TCCTATACAC TATTGCACTC CAGCTGGTTA TGCGATTCTA

7501 CAGTGTAATA ATAAGGCATT CAATGGGACA GGACCATGCC ATAATGTTAG CACGGTACAA

7561 TGTACACATG GAATTAAGCC AGTGGTATCA ACTCAACTAC TGTTAAATGG TAGCCTAGCA

7621 GAAAGAGAGA TAATAATTAG ATCTGAAAAT CTGACAAACA ATGCCAAAAC AATAATAGTA

7681 CATCTTAATG AATCTGTAGA AATTGTATGT ACAAGACCCA ACAATAATAC AAGAAAAAGT

7741 ATAAGGATAG GACCAGGACA AACATTCTAT GCAACAGGAG AAATCATAGG AGACATAAGA

7801 CAAGCACATT GTAACATTAG TGAAGATAGT TGGAATAAAA CTTTATGGAA GGTAAGTAAA

7861 AAATTAGCAG AATACTTCCC TAATAAAACA ATAAACTTTA CATCATCCTC AGGAGGGGAC

7921 CTAGAAATTA CAACACATAG CTTTAATTGT AGAGGAGAAT TTTTCTATTG TAATACATCA

7981 AAACTGTTTA ACAGTACATA CAATAGTACA ACAGGCCTGT TTAACGGTAC ATACAATAGT

8041 ACAAAAGAAG GTAATTCAAG CTCAACCATC ATACTTCCAT GCAGAATAAA GCAAATTATA

8101 AACATGTGGC AGGAGGTAGG ACGAGCAATG TATGCCCCTC CTATTGAAGG AAACATAACA

8161 TGTAAATCAA CTATCACAGG ACTATTATTG GTACGTGATG GAGGAAAGAC AAATGGTACA

8221 GAGGCAAATA GAACAGAGAT ATTCAGACCT GGAGGAGGAG ATATGAGGGA CAATTGGAGA

8281 AGTGAATTAT ATAAATATAA AGTGGTAGAA ATTAAGCCAT TGGGAGTAGC ACCCACTGCA

8341 GCAAAAAGGA GAGTGGTGGA GAGAGAAAAA AGAGCAGTGG GACTGGGAGC TGTGTTCCTT

8401 GGGTTCTTGG GAGCAGCAGG AAGCACTATG GGCGCGGCGT CAATAACGCT GACGGTACAA

8461 GCCAGACAAT TGTTGTCTGG TATAGTGCAA CAGCAAAGCA ATTTGCTGAG AGCTATAGAG

8521 GCGCAACAGC ATCTGTTGCA ACTCACGGTT TGGGGCATTA AACAGCTACA GACAAGAGTC

8581 CTGGCTATAG AAAGATACCT AAAGGATCAA CAGCTCCTAG GGATTTGGGG CTGCTCTGGA

8641 AAACTCATCT GCACTACTGC TGTACCTTGG AACTCCAGTT GGAGTAACAA GTCTTACACA

8701 GAGATTTGGG ATAACATGAC CTGGATGCAG TGGGATAAGG AAATTAGTAA TTACACAAAC

8761 ACAATCTACA GGTTGCTTGA AGACTCGCAA AACCAGCAGG AAAGAAATGA AAAAGATTTA

8821 TTGGCATTGG ACAGTTGGAA AAATCTATGG AGTTGGTTTG ACATAACAAA TTGGCTGTGG

8881 TATATAAGAA TATTCATAAT GATAGTAGGA GGCTTGATAG GTTTAAGAAT AGTTTTTGCT

8941 GTACTTTCTA TAGTAAATAG AGTTAGGCAG GGATATTCAC CATTATCGTT TCAGACCCTC

9001 CTCCCAGCCT CGAGGGGACC CGACAGGCCC GAAGGAACAG AAGAAGAAGG TGGAGAGAGA

9061 GACAGAGACA GATCCGGTCC ATCAGTGAAC GGATCCTTGG CACTTATCTG GGACGATCTG

9121 CGGAGCCTGT GCCTCTTCAG CTACCACCGC TTGAGAGACT TACTCTTGAT TGTAACGAGG

9181 ATTGTGGAAC TTCTGGGACG CAGGGGGTGG GAAGCCCTCA AATATTGGTG GAATCTCCTA

9241 CAGTATTGGA GTCAGGAACT AAAGAATAGT GCTGTTAGCT TGCTACAATA TGGGTGGAGC

9301 TATTTCCATG AGGCGGTCCA GGCCGTCTGG AGATCTGCGA CAGAGACTCT TGCGGGCGCG

9361 TGGGGAGACT TATGGGAGAC TCTTAGGAGA GGTGGAAGAT GGATACTCGC AATCCCCAGG

9421 AGGATTAGAC AAGGGCTTGA GCTCACTCTC TTGTGAGGGA CAGAAATACA ATCAGGGACA

9481 GTATATGAAT ACTCCATGGA GAAACCCAGC TGAAGAGAGA GAAAAATTAG CATACAGAAA

9541 ACAAAATATG GATGATATAG ATGAGGAAGA TGATGACTTG GTAGGGGTAT CAGTGAGGCC

9601 AAAAGTTCCC CTAAGAACAA TGAGTTACAA ATTGGCAATA GACATGTCTC ATTTTATAAA

9661 AGAAAAGGGG GGACTGGAAG GGATTTATTA CAGTGCAAGA AGACATAGAA TCTTAGACAT

9721 ATACTTAGAA AAGGAAGAAG GCATCATACC AGATTGGCAG GATTATACCT CAGGACCAGG

9781 AATTAGATAC CCAAAGACAT TTGGCTGGCT ATGGAAATTA GTCCCTGTAA ATGTATCAGA

9841 TGAGGCACAG GAGGATGAGG AGCATTATTT AATGCATCCA GCTCAAACTT CCCAGTGGGA

9901 TGACCCTTGG GGAGAGGTTC TAGCATGGAA GTTTGATCCA ACTCTGGCCT ACACTTATGA

9961 GGCATATGTT AGATACCCAG AAGAGTTTGG AAGCAAGTCA GGCCTGTCAG AGGAAGAGGT

10021 TAGAAGAAGG CTAACCGCAA GAGGCCTTCT TAACATGGCT GACAAGAAGG AAACTCGCTG

10081 AAACAGCAGG GACTTTCCAC AAGGGGATGT TACGGGGAGG TACTGGGGAG GAGCCGGTCG

10141 GGAACGCCCA CTTTCTTGAT GTATAAATAT CACTGCATTT CGCTCTGTAT TCAGTCGCTC

10201 TGCGGAGAGG CTGGCAGATT GAGCCCTGGG AGGTTCTCTC CAGCACTAGC AGGTAGAGCC

10261 TGGGTGTTCC CTGCTAGACT CTCACCAGCA CTTGGCCGGT GCTGGGCAGA GTGACTCCAC

10321 GCTTGTTTGC TTAAAGCCCT CTTCAATAAA GCTGCCATTT TAGAAGTAAG CTAGTGTGTG

10381 TTCCCATCTC TCCTAGCCGC CGCCTGGTCA ACTCGGTACG CGGCCGcgtg atacgcctat

10441 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg

10501 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc

10561 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta

10621 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg

10681 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg

10741 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac

10801 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg

10861 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt

10921 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg

10981 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac

11041 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt

11101 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag

11161 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc

11221 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc

11281 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta

11341 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg

11401 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga

11461 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac

11521 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa

11581 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat

11641 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc

11701 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg

11761 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc

11821 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg

11881 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg

11941 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa

12001 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg

12061 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacaggagagcgcacga

12121 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct

12181 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca

12241 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc

12301 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg

12361 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc

12421 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc

12481 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg

12541 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc

12601 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg

12661 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc

12721 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt

12781 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc

12841 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc

12901 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact

12961 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac

13021 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca

13081 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag

13141 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt

13201 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag

13261 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg

13321 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat

13381 ggatatgttc tgccaagggt tggtttgc

Claims (10)

1. the structure of a SHIV infections clone, it is characterized in that this SHIV infections clone SHIV-KBQJ-12 chimeric the env gene of Chinese HIV-1 Major Epidemic strain CRF08_BC, it derives from the infections clone strain QJ001 of HIV-1CRF08_BC, the whole intracellular region, cross-film district and the part extracellular region that comprise gp120 and gp41, the position of replaced env gene of entering is 6103-8249 corresponding to the nucleotide position of canonical reference strain HXB2; The full-length gene group sequence of QJ001 is carried out evolutionary analysis show that QJ001 belongs to the CRF08_BC hypotype, equally the env gene that is entered SHIV-KBQJ-12 by new displacement is carried out evolutionary analysis and show that SHIV-KBQJ-12 belongs to the HIV-1CRF08_BC hypotype;

Described HIV-1QJ001 strain preserving number is CCTCC NO:M2013066, and described SHIV-KBQJ-12 strain preserving number is CCTCC NO:M2013067.

2. the structure of SHIV infections clone according to claim 1 is characterized in that the structure employing of described SHIV-KBQJ-12 infections clone comprises the steps:

(1) employing has the plasmid pBR322 of low copy number as carrier, utilize 3 '-SHIV-KB9 and 5 ' that HIV/AIDS Reagent Program provides-SHIV-KB9 plasmid amplification to go out the full-length gene group of SHIV-KB9, and connect in the pBR322 carrier, obtain in STBL3E.coli, stablizing the SHIV-KB9/pBR322 infective molecule cloning that copies, and with this genome skeleton as structure SHIV-KBQJ-12;

(2) utilize overlap extension pcr, take SHIV-KB9/pBR322 and HIV-1QJ001 as template, increase and enzyme cuts back to close and obtains Sph I-Pst I and Pst I-two gene fragments of Xho I with corresponding primer; Again Sph I-Pst I is connected into and obtain subclone pKBQJ_SPs among the carrier pGEM5Zf (-), Pst I-Xho I is connected into obtain subclone pKBQJ_PsX among the carrier pBluescriptSK (-); The grand pKBQJ_SPs in the Asia that will obtain at last and pKBQJ_PsX are respectively after sequence verification is correct, with SphI and PstI, PstI and XhoI, SphI and XhoI difference double digestion pKBQJ_SPs, pKBQJ_PsX and SHIV-KB9/pBR322, glue connects, transforms the correct mono-clonal of STBL3E.coli picking after reclaiming required gene fragment can obtain described SHIV-KBQJ-12.

3. the structure of SHIV infections clone according to claim 2, adopt following step when it is characterized in that making up infective molecule cloning SHIV-KB9/pBR322:

(1) take carrier pBR322 as template, utilize primer EE_EcoA and primer EE_Eag_B to carry out pcr amplification, the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min; Then sepharose reclaims pcr amplification product, with these two enzymes of EcoRI and EagI the gene product of recovery is carried out behind the double digestion agarose gel electrophoresis again and separates enzyme and cut product;

(2) take plasmid 5 '-SHIV-KB9 as template, utilize primer KB_U5Eco_A and primer KB_pbs_B to carry out pcr amplification, the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min; Then sepharose reclaims pcr amplification product, with these two enzymes of EcoRI and NarI the gene product of recovery is carried out behind the double digestion agarose gel electrophoresis again and separates enzyme and cut product;

(3) plasmid 5 '-SHIV-KB9 is carried out double digestion with NarI and SphI after, separate the gene fragment that enzyme is cut the about 5.6Kb of product by agarose gel electrophoresis;

Equally, plasmid 3 '-SHIV-KB9 carried out double digestion with SphI and NotI after, separate the gene fragment that enzyme is cut the about 4Kb of product by agarose gel electrophoresis;

(4) four gene fragments with above-mentioned recovery connect, and transform the STBL3E.coli competent cell, can correctly be cloned SHIV-KB9/pBR322;

Wherein, the sequence of primer is:

EE_EcoA:5’-TGT GAA TTC GCA AAC CAA CCC TTG GCA G-3’

EE_Eag_B:5’-AAA CGG CCG CGT GAT ACG CCT ATT-3’

KB_U5Eco_A:5’-AGC GAA TTC TAG ACA TAT ACT TAG AAA AGG AAG-3’

KB_pbs_B:5’-TGT TCA GGC GCC AAT CTG CTA G-3’。

4. the structure of SHIV infections clone according to claim 2 is characterized in that obtaining adopting following steps to make up the SHIV-KBQJ-12 infective molecule cloning after the genome skeleton SHIV-KB9/pBR322:

(1) for obtaining the SphI-PstI fragment, at first take SHIV-KB9/pBR322 as template, carry out first round pcr amplification with primer tatSph_A and envSU_B, this pcr amplification reaction condition is 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take QJ001 as template, carry out first round pcr amplification with primer vpuPac_A and envCS_B simultaneously, this pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Then take two pcr amplification products of the first round as template, carry out second with primer tatSph_A and envCS_B and take turns pcr amplification, second takes turns the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take turns amplified production with SphI and PstI double digestion second again, and connect among the carrier pGEM5Zf (-), obtain subclone pKBQJ_SPs;

(2) for obtaining the PstI-XhoI fragment, at first take SHIV-KB9/pBR322 as template, carry out first round pcr amplification with primer envECD_A and SIVnef_B, this pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take QJ001 as template, carry out first round pcr amplification with primer envCS_A and envECD_B simultaneously, this pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Then take two pcr amplification products of the first round as template, carry out second with primer envCS_A and SIVnef_B and take turns amplification, second takes turns the pcr amplification reaction condition is: 94 ℃ of 2min; 94 ℃ of 15s, 55 ℃ of 15s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 15min;

Take turns amplified production with PstI and XhoI double digestion second again, and connect among the carrier pBluescriptSK (-), obtain subclone pKBQJ_PsX;

(3) with the subclone pKBQJ_SPs that obtains and pKBQJ_PsX respectively after sequence verification is correct, with SphI and PstI, PstI and XhoI, SphI and XhoI difference double digestion pKBQJ_SPs, pKBQJ_PsX and SHIV-KB9/pBR322, glue connects and conversion STBL3E.coli competent cell after reclaiming required gene fragment, can correctly be cloned SHIV-KBQJ-12;

Wherein, the sequence of primer is:

tatSph_A:5’-TTG GCA TGC TGT AGA GCA AGA AAT GGA GCC AGT AG-3’

envSU_B:5’-CAG GTA CCC CAT AAT AGA CTG-3’

vpuPac_A:5’-TGG TTA ATT AAA AGA ATT AGG GAA AGA GCA G-3’

envCS_B:5’-GCC CAT AGT GCT TCC TGC TGC TCC-3’

envECD_A:5’-ATT CAT AAT GAT AGT AGG AGG-3’

SIVnef_B:5’-AAG AGT CAC TGT CGC AGA TCT CC-3’

envCS_A:5’-ATA TGA GGG ACA ATT GGA GAA GTG-3’

envECD_B:5’-AAA GGT GAG TAT CCC TGC CTA ACT C-3’。

5. the structure of SHIV infections clone according to claim 1, when it is characterized in that preparing SHIV-KBQJ-12 virus, use the Lipofectamine2000 transfection reagent that nucleotide sequence is confirmed errorless SHIV-KBQJ-12 plasmid transfection 293T cell, collect viral supernatant after 48 hours in transfection and get final product.

6. the structure of SHIV infections clone according to claim 1, it is characterized in that after the SHIV-KBQJ-12 virus transfection 293T clone, collecting cell is made ultrathin section(ing), under transmission electron microscope, can observe SHIV-KBQJ-12 and in the 293T cell, successfully assemble out complete virion, show each gene of SHIV-KBQJ-12 embedded virus succeeded express and display function.

7. the structure of SHIV infections clone according to claim 1, it is characterized in that utilizing two kinds of clones of GHOST-CD4-CXCR4 and GHOST-CD4-CCR5, detect the cell quantity of the expression GFP that is excited in the Ghost cell of expressing different accessory receptors by flow cytometer, thereby determine SHIV-KBQJ-12 virus and be with CCR5 as accessory receptor, have the characteristic of scavenger cell preferendum.

8. the structure of SHIV infections clone according to claim 1 is characterized in that adopting following detection method to judge the replication of SHIV-KBQJ-12 virus in people or rhesus monkey lymphocyte:

With 500ng SHIV-KBQJ-12 virus infection with PHA stimulate 72 hours 4 * 10 ⁶The PBMC cell of people or rhesus monkey, 37 ℃, 5%CO ₂Overnight incubation, removed viral supernatant liquor at the 0th day, and with the 1640 culture medium culturing cells that contain 25U/ml IL-2 and 10% foetal calf serum, then infecting the 3rd, 6,9,12,15,18,21 day, collect the supernatant sample, detect the use of SIV p27 antigen in-80 ℃ of frozen preparing against after the packing, and the adjustment cell concn is 2 * 10 ⁶Individual/ml, continue to cultivate with 1640 substratum that contain 25U/ml IL-2 and 10% foetal calf serum;

Draw by the p27 expressing quantity that is determined at the supernatant sample that above-mentioned different time points collects and to infect curve, can learn that SHIV-KBQJ-12 virus can both carry out copying of toxigenicity in the PBMC of people and rhesus monkey cell.

9. the structure of SHIV infections clone according to claim 1 is characterized in that adopting following detection method to judge the biological characteristics of SHIV-KBQJ-12 virus in the rhesus monkey body:

The SHIV-KBQJ-12 virus of 6000ng is inoculated two Health China rhesus monkeies in the intravenous mode of hind leg, and then gather rhesus monkey vein EDTA anticoagulation by following time point: first month gathers weekly 2 times, gathers once weekly afterwards; With above-mentioned extracting genome DNA in the rhesus monkey vein EDTA anticoagulation that different time points gathers out, and be that the specific fragment of 477bp increases to the Gag section length, to confirm whether whether SHIV-KBQJ-12 virus has been incorporated in the rhesus monkey genome, set up productive infection in the monkey body;

Behind the virus inoculation 98 days, above-mentioned two rhesus monkeies are practised mercy killing, gather various tissues, extract DNA and RNA in each tissue, Gag section length with RNA and DNA in each tissue of method detection of pcr amplification is the specific fragment of 477bp, with the infectivity of further confirmation SHIV-KBQJ-12 virus to rhesus monkey;

The result shows that above-mentioned two rhesus monkeies have begun namely to have set up stable productive infection at the 6th day and the 12nd day respectively, and viral genome also is incorporated in the rhesus monkey genome; SHIV-KBQJ-12 virus mainly is distributed in reproductive system, central nervous system and unifies in the gut associated lymphoid tissue.

10. the application of the described SHIV infections clone of arbitrary claim among the claim 1-9 is characterized in that described SHIV-KBQJ-12 infections clone is used for setting up SHIV/ China rhesus monkey infection model.

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