CN107226848A - Dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method and application - Google Patents

Dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method and application Download PDF

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CN107226848A
CN107226848A CN201710322563.6A CN201710322563A CN107226848A CN 107226848 A CN107226848 A CN 107226848A CN 201710322563 A CN201710322563 A CN 201710322563A CN 107226848 A CN107226848 A CN 107226848A
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dengue
protein
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truncated segment
pqe30
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王明连
陈基湘
刘强强
李劲涛
张婷
钟儒刚
刘伟
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Beijing University of Technology
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Abstract

The present invention provides a kind of dengue 2-type virus NS5 PROTEIN Cs end truncated segment, and it is fragment (the SEQ ID NO for the NS5 PROTEIN Cs end 70kDa sizes of DENV 2 expressed using prokaryotic expression system (E.Coli):1) RdRp active structure domains, are contained, viral RNA genes group is replicated and played a crucial role, are expected to turn into the target for blocking or suppressing virus multiplication.ELISA experiments show, the dengue virus infection person serum of the truncated segment and four types can produce positive reaction, reactivity has significant difference with healthy population, it thus can be used for the immunology diagnosis of dengue virus infection, and a reaction can detect the virus infection of four types, detection time is saved, blood sample consumption is few.Thus the quick and precisely diagnosis for dengue virus infection is provided a kind of effective way by the present invention, is that the timely treatment of patient is raced against time, and improves cure rate.

Description

Dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method and application
Technical field
The present invention relates to genetic engineering and virological immunology diagnostic field, specifically, it is related to a kind of dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method and application.
Background technology
The World Health Organization (WHO) data display, the past about seven during the decade, dengue fever (Dengue fever, DF) is As epidemic situation, mosquito matchmaker with fastest developing speed infects disease, jeopardizes throughout the year and lives in the torrid zone, 128, subtropical zone countries and regions about 20 The life security of~50 hundred million people.The DF incidences of disease had increased sharply more than 30 times in past 40 years, in the world in rising situation, and And just continually these non-lesion diffusions toward the U.S., southern Europe and Australia.It is annual to there are 5,000 ten thousand to 100,000,000 people to infect dengue virus (Dengue virus, DENV).After DENV infection human bodies self limiting DF and clinical symptoms more serious Dengue can be caused to go out Blood-head (Dengue hemorrhagic fever, DHF) and dengue shock syndrome (Dengue shock syndrome, DSS), then both cause about 50,000 people's hospitalizations every year, and the death rate is 2.5%.The state of an illness of most dengue fevers is all Compare light.But there are about 10% patient can develop into serious DHF/DSS, have small part patient even can be because of blood vessel in addition The too high bleeding of permeability and it is dead.
Even think that dengue virus there are four types at present, people can be infected, wherein 2 type severe rates and the death rate are above Other types.Primary infection of the crowd to any type DENV is more sensitive, and it is more lasting to homologous virus to be obtained after infection Immunity, is typically kept for 1~4 year, and the immunity to special-shaped virus is then very of short duration, only maintains 2~12 months.Thus infection one It may also happen that secondary or consecutive infection is secondary infection after individual type DENV, and Secondary cases abnormal shape infection is likely to cause DHF/DSS, so as to trigger the at a relatively high incidence of disease and case fatality rate.Its mechanism is the infection enhancing of antibody-dependant (Antibody dependent enhancement, ADE) effect, i.e. intersection pre-existing in Secondary cases abnormal shape infection resist Body can promote virus to monocyte, huge with virus combination by the interaction of antibody and the Fc acceptors of target cells The infection of phagocyte and maturation DC cells, and then cause pachyemia, bleeding or the even clinical symptoms such as shock of having blood in stool.
On December 9th, 2015, first, the whole world chimeric yellow fever dengue fever of Sai Nuofei Pasteurs research and development is looked forward to by French medicine Tetravalent vaccine is listed in Mexico.The vaccine is come using a kind of infection clones from yellow fever 17D live attenuated vaccine virus Build four kinds of embedded viruses, each virus only envelope protein (E protein) of one of four kinds of serotype dengue virus of expression and before Memebrane protein (prM/M albumen).This serious disease for threatening whole world tens of millions of people health will be curbed, and people seem to see Dawn is arrived.But Malaysia Sarawak states have broken out an Epidemic Situation of Dengue Fever, Texas ,Usa University Hospital in 2007 The virologist Nikos at center (University of Texas Medical Branch in Galveston) Vasilakis through genome sequencing find this be one plant of newfound dengue virus of new dengue virus in crowd it is popular Possibility will cause ADE effects, so people still hold the prudent attitude to the vaccine.
The knot for being directed to type virus is produced in DENV infected individuals in the studies have shown that primary infection person body of Antibody types In the antibody monoid of structure albumen and non-structural protein, the type specificity neutralizing antibody ratio in the highest flight, and concentrates on E protein Epitope.And in secondary infection, chiasma type antibody occupies main status, and it concentrates on prM protein regions for expression is more Domain.Similarly, also there is extensive intersect in Secondary cases abnormal shape infection in the reaction of dengue virus specific T cell.Thus It was found that ADE effects are how relevant with the structural proteins E and prM of virus, and non-structural protein may not produce ADE effects.
Anti- DENV medicines are there is no at present, and metainfective quick diagnosis and symptomatic treatment are particularly important.Prevention and control DENV Propagation rely primarily on mosquito matchmaker control, can only symptomatic treatment for DF and DHF.According to national health and Family Planning Committee 《Dengue fever practice guidelines (second edition in 2014)》, " according to epidemiological history, clinical manifestation and laboratory examination results, can make The diagnosis of dengue fever.In the case where epidemiological history is unknown, made according to clinical manifestation, auxiliary examination and laboratory detection result Go out diagnosis." at present, the conventional several detection DF in laboratory method has real-time PCR detection DENV nucleic acid, using immune layer Analysis method detects DENV specificity NS1 antigens and IgM/IgG antibody etc..From patient is made a definite diagnosis in the different course of disease nucleic acid in blood serum of DF, NS1 Each index variation tendency of positive rate of antigen and IgM/IgG antibody is visible, and existing any single lab index detection is not It is enough to provide effective, reliable diagnosis, it is necessary to which two or more indexs, which are shared, just has reliability, when this undoubtedly delays diagnosis Between, add testing cost.Because DF treatment method there is no parting for various DENV, all simply symptomatic treatments, thus it is real Its existing quick diagnosis is particularly important.DENV quick diagnosis not only facilitates control epidemic situation, and can give patient couple Disease supportive treatment, is conducive to disease recovery, reduces DHF/DSS generation, reduces the death rate.
DENV belongs to flaviviridae Flavivirus, is a kind of tunicary single stranded positive-sense RNA virus, in icosahedron knot Structure, 45~55nm of diameter of virion, Genome Size is 10.7kb, 3 kinds of structural proteins of coding and 7 kinds of non-structural proteins (nonstructural protein, NS), wherein, DENV NS5 are that maximum is also most conservative in all Flavivirus albumen Multifunctional protein, and RNA polymerase (the RNA dependent that there is C-terminal (amino acid sequence is 272~900) RNA to rely on RNA polymerase, RdRp) activity, it is responsible for synthesizing viral RNA with de novo formation mechanism, in viral genome reproduction process Play central role.
The content of the invention
It is an object of the invention to provide a kind of dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method thereof.
It is a further object of the present invention to provide the dengue 2-type virus NS5 PROTEIN Cs end truncated segment in various dengue virus Application in the quick diagnosis and auxiliary diagnosis of infection.
In order to realize the object of the invention, the dengue 2-type virus NS5 PROTEIN Cs end truncated segment that the present invention is provided, its amino acid Sequence such as SEQ ID NO:Shown in 1, or the sequence is one or several amino acids formed with equal through replacing, lacking or add The amino acid sequence of function.
The truncated segment contains RdRp active structure domains, and viral RNA genes group is replicated and played a crucial role, is expected to turn into Block or suppress the target of virus multiplication.
The present inventor's early stage has been attempted using prokaryotic expression system (E.Coli) progress NS5 full length gene expression, but due to NS5 molecular weight of albumen excessive (about 104kDa) and e. coli codon preferences, it expresses more difficult, and expression quantity is relative It is relatively low, it is unfavorable for the popularization and application in future.Therefore this experiment interception NS5 genes 3 ' hold 2/3 to rebuild carrier, expression NS5C ends 70kDa (i.e. NS5/C70) polypeptide, while induced expression condition is optimized, the final soluble table for obtaining recombinant polypeptide NS5/C70 Reach.
The present invention also provides the truncated segment recombinant polypeptide NS5/C70 containing His labels, can make as follows It is standby, comprise the following steps：
(1) recombinant plasmid pQE30-NS5/C70 structure：With the plasmid containing dengue 2-type virus NS5 protein coding genes For template, primer is designed, enters performing PCR amplification, obtained amplified production BamH I, Hind III double digestions are reclaimed after purification Small fragment (about 1.9kb) is connected with linear carrier pQE30, obtains recombinant plasmid pQE30-NS5/C70；
(2) structure of prokaryotic expression system：Recombinant plasmid pQE30-NS5/C70 is transformed into E.Coli competence M15 In [pREP4], recombinant bacterium M15/pQE30-NS5/C70 is obtained；
(3) recombinant polypeptide NS5/C70 expression and purifying.
Wherein, the primer of step (1) is：
- the GG of forward primer F1 5 'GGATCC(underscore is BamH I digestions to CCAAACCTAGACATAATCGGGAAA-3 ' Site)
- the GC of reverse primer R1 5 'AAGCTT(underscore is Hind III digestions position to CTACCACAGGACTCCTGCCT-3 ' Point)
Step (3) is specially：
1. Zengjing Granule stage：Recombinant bacterium M15/pQE30-NS5/C70 is inoculated in 2 × YT cultures containing Amp and Kana In base, the glucose of addition 1% is cultivated to OD under 180rpm, 37 DEG C of aeration conditions570For 0.7-0.8.
2. induction period：IPTG to final concentration of 1mM is added, in 25 DEG C, 180rpm is induced, respectively at 0h, 1h, 2h, 3h, 4h, 5h are sampled, and are carried out 10%SDS-PAGE analyses and are shown that 4h is the optimal induction time of NS5/C70 polypeptides.
3. supernatant is collected by centrifugation and with 0.45 μm of membrane filtration；Filtrate is collected with nickel post (Ni2+- NTA-Agarose posts) parent And chromatographic purifying, then the imidazole solution gradient elution through various concentrations (10mM, 20mM, 40mM, 250mM, 500mM), final To the NS5/C70 recombinant polypeptides of purifying.
In previous experiments, inventor attempts to optimize expression vector, engineering bacteria, expression condition.Due to the albumen Fragment is larger (70kDa), and the expression label of selection should be as small as possible, and His labels are current minimum labels, and immunogenicity is relative It is relatively low, can be by the albumen direct injection animal body of purifying, passing immunization.In view of carrier pQE30 initiation codon Carry 6 × His labels between AUG and restriction enzyme site BamH I/Hind III, and Host Strains M15 and plasmid pQE30 are more Match somebody with somebody.If using XL1Blue instead as engineering bacterium expression polypeptide, expression quantity is poor compared with M15.
Recombinant polypeptide NS5/C70 and four kinds of serotype dengue fever patients serums are reacted, recombinant polypeptide DENV-2NS5/ is used C70 is reacted with four serotype dengue fever patients serums as the hole elisa Plates of antigen coat 96, and with not infected normal person Serum does negative control.As a result it is 1 in serum dilution to show the recombinant peptide:500~1:When 16000, not only feel with DENV-2 Dye person's serum shows strong positive reaction, other three type dengue virus infection person serum is also presented with positive or strong positive anti- Answering property (Fig. 5).
The present invention also provides the dengue 2-type virus NS5 PROTEIN Cs end truncated segment and prepared for different serotypes Dengue Application in the immunological diagnostic reagent of virus infection.
The present invention is also provided to be stepped on by prepared by dengue 2-type virus NS5 PROTEIN Cs end truncated segment for different serotypes The immunological diagnostic reagent of leather virus infection.
The present invention also provides the dengue 2-type virus NS5 PROTEIN Cs end truncated segment and prepared for different serotypes Dengue Application in the colloidal gold immunochromatographimethod diagnosis test paper of virus infection.
The present invention also provides the colloidal gold immunochromatographimethod diagnosis test paper for dengue virus infection, and the test strips are by sample Product pad, colloidal gold pad, NC films, absorbent filter and PVC bottom plates composition, sample pad, colloidal gold pad, NC films and absorbent filter phase successively Mutually superposition is pasted onto on PVC bottom plates.
Wherein, the anti-colloidal gold composite of goat-anti people two is coated with colloidal gold pad；NC films are provided with p-wire and Quality Control Line, the p-wire is fixed with the dengue 2-type virus NS5 PROTEIN Cs end truncated segment or the recombinant polypeptide containing His labels, The nature controlling line is fixed with SPA (staphylococcal protein A).
The present invention further provides be used for various dengue virus sense containing above-mentioned diagnostic reagent or above-mentioned diagnosis test paper The quick detection kit of dye.
The DENV-2NS5 PROTEIN Cs end 70kDa fragments that the present invention is expressed using prokaryotic expression system (E.Coli) first, bag RdRp active structure domains are contained, viral RNA genes group are replicated and played a crucial role, be expected to turn into blocking or suppress virus multiplication Target.It is the present invention relates to the optimization of recombinant polypeptide expression condition and affine pure using the 6 × His-Tag and nickel post of polypeptide N-terminal Change the technological process of the polypeptide, and with the restructuring NS5/C70 polypeptides obtained as antigen, with four kinds of serotype dengue fever patient's blood Clearance response, negative control is done with not infected normal human serum, as a result shows that the recombinant products can be with four kinds of serotype diseases Positive reaction is presented in human serum, thus the recombinant products can be used in the immunology diagnosis of dengue virus infection, and one anti- The virus infection of four types should can be detected, detection time is saved, blood sample consumption is few.Thus the present invention will be to step on The quick and precisely diagnosis of leather virus infection provides a kind of effective way, is that the timely treatment of patient is raced against time, improves cure rate.
Brief description of the drawings
Fig. 1 identifies electrophoretogram for the plasmid pQE30-NS5/C70 of the embodiment of the present invention 1 single, double digestion；Wherein, 1,2：Matter Grain pQE30-NS5/C70；3、4：BamH I, Hind III double digestions plasmid product (upper 3.4kb, lower 1.9kb)；5、6：BamH I Single endonuclease digestion plasmid product (5.3kb)；7、8：Hind III single endonuclease digestions plasmid products (5.3kb).
Fig. 2 is the 10%SDS- that 1DENV-2NS5/C70 recombinant polypeptide induced expression amounts of the embodiment of the present invention are changed over time PAGE electrophoresis results；Wherein, plasmid-free：M15 bacterial strains (are free of pQE30-NS5/C70).
Fig. 3 is the 10%SDS-PAGE electrophoresis knots of the various concentrations imidazole solution gradient elution filtered solution of the embodiment of the present invention 1 Really；Wherein, 1：M15 bacterial strains (are free of pQE30-NS5/C70)；2、3：Lysate supernatant；4-13 is imidazoles liquid eluent (4-6: 10mM, 20mM, 40mM；7-11：250mM；12-13：500mM)；14：M15/pQE30-NS5/C70(IPTG 0mM).
Fig. 4 is that the recombinant polypeptide NS5/C70 of the embodiment of the present invention 2 and four serotype dengue fever patients and normal human serum are anti- Ying Xing；Wherein, "+" represents reacting positive, and criterion is that OD is more than or equal to healthy population OD averages+1.96SE；" ++ " table Show strong positive, criterion is that OD is more than or equal to healthy population OD averages+2.58SE。SEFor standard error.
Fig. 5 is that the ELISA experiments of dengue virus infection person's serum of recombinant polypeptide NS5/C70 of the present invention and four types are tied Really.
Fig. 6 is the colloidal gold immunochromatographimethod quick diagnosis test strips structure schematic diagram of the embodiment of the present invention 3.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
Dengue 2-type virus NS5 PROTEIN Cs end truncated segment of embodiment 1 and preparation method thereof
The dengue 2-type virus NS5 PROTEIN Cs end truncated segment that the present embodiment is provided, size about 70kDa, fragment is contained RdRp active structure domains, amino acid sequence such as SEQ ID NO:Shown in 1.
The recombinant polypeptide NS5/C70 preparation methods of truncated segment of the N-terminal with His labels are as follows：
1st, recombinant plasmid pQE30-NS5/C70 structure：With the plasmid containing dengue 2-type virus NS5 protein coding genes For template, primer is designed, enters performing PCR amplification, obtained amplified production BamH I, Hind III double digestions are reclaimed after purification Small fragment (about 1.9kb) is connected with linear carrier pQE30, obtains recombinant plasmid pQE30-NS5/C70, and its digestion qualification result is shown in Fig. 1.
The primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and sequence is as follows：
- the GG of forward primer F1 5 'GGATCC(underscore is BamH I digestions to CCAAACCTAGACATAATCGGGAAA-3 ' Site)
- the GC of reverse primer R1 5 'AAGCTT(underscore is Hind III digestions position to CTACCACAGGACTCCTGCCT-3 ' Point)
2nd, the structure of prokaryotic expression system：Recombinant plasmid pQE30-NS5/C70 is transformed into E.Coli competence M15 In [pREP4], recombinant bacterium M15/pQE30-NS5/C70 is obtained.
3rd, recombinant polypeptide NS5/C70 expression and purifying：
1. Zengjing Granule stage：Recombinant bacterium M15/pQE30-NS5/C70 is inoculated in 2 × YT culture mediums (Amp+、Kana+) In, the glucose of addition 1% is cultivated to OD under 180rpm, 37 DEG C of aeration conditions570For 0.7-0.8.
2. induction period：IPTG to final concentration of 1mM is added, in 25 DEG C, 180rpm is induced, respectively at 0h, 1h, 2h, 3h, 4h, 5h are sampled, and are carried out 10%SDS-PAGE analyses and are shown that 4h is the optimal induction time (Fig. 2) of NS5/C70 polypeptides.
3. supernatant is collected by centrifugation and with 0.45 μm of membrane filtration；Collect filtrate and use Ni2+- NTA-Agarose post affinity chromatographys Purifying, then the imidazole solution gradient elution through various concentrations (10mM, 20mM, 40mM, 250mM, 500mM), finally give purifying NS5/C70 recombinant polypeptides.Imidazoles liquid (such as 10mM, 20mM, the 40mM) elution of wherein low concentration removes desired polypeptides (about 70kDa) Outside foreign protein, and the imidazoles liquid of high concentration (i.e. 250mM) mainly elutes desired polypeptides NS5/C70 (Fig. 3).
The diagnostic application of the dengue 2-type virus NS5 PROTEIN Cs end truncated segment of embodiment 2
Comprise the following steps that：
1st, envelope antigen：It is coated with using recombinant polypeptide NS5/C70 as antigen, the albumen being stored in -20 DEG C is taken out, put Enter micro- from coating diluted antigen to concentration 100ng/100 μ L, with sealed membrane sealing plank, 4 DEG C of coatings in centrifuge Overnight.
2nd, wash：Liquid in coating plate overnight is exhausted and patted dry, 200 μ LELISA cleaning solutions of addition, which are placed in, per hole shakes Bed 1min, suctions out and cleaning solution and is patted dry in hole.Repeat 4~5 times.
3rd, close：200 μ L ELISA confining liquids, 37 DEG C of incubation 2h are added per hole.
4th, wash：Liquid in the plate of closing is exhausted and patted dry, 200 μ LELISA cleaning solutions are added per hole and are placed in shaking table 1min, suctions out and cleaning solution and is patted dry in hole.Repeat 4~5 times.
5th, -20 DEG C of refrigerators take out blood serum sample, melt under the conditions of being placed in 4 DEG C, and each blood serum sample sets multiple holes 2, often Negative blood control multiple holes and each 2 of blank control are set in individual 96 orifice plate.
6th, test serum, negative serum (PBS groups), dummy is incubated：Test serum sample is entered with containing PBS Row dilutes, and extension rate is respectively：1:500,1:1000,1:2000,1:4000,1:8000,1:16000 every holes, which are added, to be diluted The μ L of test serum sample 100, negative hole add negative serum, blank control wells add equivalent dilution, 37 DEG C incubation 1h.
7th, washing is with step 2.
8th, secondary antibody is incubated：Secondary antibody is Goat anti human IgG, dilutes 4000 times with 10%FBS PBS, secondary antibody 100 is added per hole μ L, 37 DEG C of incubation 1h.
9th, washing is with step 2.
10th, develop the color：TMB 100 μ L, 37 DEG C of 10~15min of standing, then the μ of addition ELISA terminate liquids 50 per hole are added per hole L。
11st, 96 orifice plate ELISA Plates are placed in ELIASA, 450nm wavelength reads OD values.
With recombinant polypeptide DENV-2NS5/C70 as the hole elisa Plates of antigen coat 96, with four serotype dengue fever patients Seroreaction, and compared with healthy population serum, as a result show, polypeptide NS5/C70 not only has to DENV-2 immune serums There is good reactivity, and also there is good reactivity to other three kinds of serotypes.The recombinant polypeptide is in serum dilution For 1:500~1:Positive (+) or strong positive (++) reaction (Fig. 4) is generated to the infected person anteserum of four types when 16000. The recombinant products respectively with the OD curves of dengue virus infection person's serum ELISA reactions of four types also all with normal healthy controls people Group has significant difference (Fig. 5).
Embodiment 3 prepares colloidal gold immunochromatographimethod quick diagnosis test strips
Specific antibody is fixed on film with ribbon, colloid gold label reagent (antibody or monoclonal antibody) absorption On pad, after serum to be checked (or dilute serum) is added in the sample pad of test strips one end, by capillarity forward It is mobile, when being reacted to each other after the colloid gold label reagent on dissolving pad, then being moved to the region of fixed antigen or antibody, The conjugate of serum and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and is gathered in detection band, can pass through It is observed visually colour developing result.This method has developed into diagnosis test paper, is particularly suitable for use in field diagnostic and quick diagnosis.Will The recombinant protein product of the present invention is prepared into colloid gold test paper, is conveniently used for the quick diagnosis of dengue virus infection.
Cleaning Principle：
Indirect method is generally used for detecting antibody, and approximate with double antibody sandwich method principle, difference is, with test antibodies Corresponding antigen protein coating detection line (T lines), colloidal gold pad is soaked with the corresponding collaurum mark secondary antibody of test antibodies, with can be with The staphylococcal protein A (SPA) of antibody binding is fixed on nature controlling line (C lines).In detection process, such as one positive sample, Antibody in sample to be tested is first combined with gold labeling antibody, by T lines during coated antigen, test antibodies and antigen binding, no Disconnected accumulation collaurum is until send color signal.Unnecessary gold labeling antibody is combined when chromatographing to C lines with the SPA of nature controlling line, accumulation After develop the color.The positive findings presented is T lines and the double colour developings of C lines；Negative findings is that T lines do not develop the color, the colour developing of C lines；C lines do not develop the color It is then null result.
Concrete scheme：
The present embodiment provide the colloidal gold immunochromatographimethod diagnosis test paper for dengue virus infection, the test strips by Sample pad, colloidal gold pad, NC films, absorbent filter and PVC bottom plates composition, sample pad, colloidal gold pad, NC films and absorbent filter are successively It is overlapped mutually and is pasted onto on PVC bottom plates (Fig. 6).
Wherein, the anti-colloidal gold composite of goat-anti people two is coated with colloidal gold pad；NC films are provided with p-wire and Quality Control Line, the p-wire is fixed with dengue 2-type virus NS5 PROTEIN Cs end truncated segment described in claim 1 or containing His labels Recombinant polypeptide, the nature controlling line is fixed with SPA.
As a result illustrate：
(1) it is positive：Successively there is red response line in test-strips upper and lower ends.
Under the traction of absorbent material, test serum is moved up in strip, and (goat-anti people resists with gold mark secondary antibody first Body) antigen antibody complex is combined to form, it is many with coated restructuring on p-wire when antigen antibody complex is up to p-wire Peptide is combined, and constantly accumulation colloid is until send color signal.Unnecessary gold mark goat anti-human antibody chromatograph to during nature controlling line with coating Combine, developed the color after accumulation in the SPA of nature controlling line.The positive findings presented is the double colour developings of T line C lines.
(2) it is negative：Test-strips upper end only has a red nature controlling line and occurred.
Only have gold labeling antibody up without measured antibody in test serum, in test-strips and combined with the SPA at nature controlling line, made Gold grain deposition is aobvious red here.
(3) it is invalid：Nature controlling line does not develop the color, and shows test failure or test-strips failure.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing University of Technology
<120>Dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method and application
<130> KHP171112550.3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 629
<212> PRT
<213>Dengue-2 Virus 43 Strain Isolated
<400> 1
Pro Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
1 5 10 15
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp
20 25 30
Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser
35 40 45
Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Val
50 55 60
Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln
65 70 75 80
Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Gln Glu Pro Lys
85 90 95
Glu Gly Thr Lys Lys Leu Met Lys Ile Thr Ala Glu Trp Leu Trp Lys
100 105 110
Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys Thr Arg Glu Glu Phe
115 120 125
Thr Arg Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp
130 135 140
Glu Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu Asp Ser Arg Phe
145 150 155 160
Trp Glu Leu Val Asp Lys Glu Arg Asn Leu His Leu Glu Gly Lys Cys
165 170 175
Glu Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly
180 185 190
Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu
195 200 205
Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp
210 215 220
His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly
225 230 235 240
Leu Tyr Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
245 250 255
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr
260 265 270
Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met Glu Gly
275 280 285
Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn
290 295 300
Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly Thr Val Met Asp
305 310 315 320
Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr
325 330 335
Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu Ile Arg Gln Met
340 345 350
Glu Gly Glu Gly Val Phe Lys Ser Ile Gln His Leu Thr Val Thr Glu
355 360 365
Glu Ile Ala Val Gln Asn Trp Leu Ala Arg Val Arg Arg Glu Arg Leu
370 375 380
Ser Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro Leu Asp
385 390 395 400
Asp Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn Asp Met Gly Lys Val
405 410 415
Arg Lys Asp Ile Gln Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp
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Asp Gly Arg Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile
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Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr
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Ala Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
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His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val
500 505 510
Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala
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Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn Arg
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<212> DNA
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ggggatcccc aaacctagac ataatcggga aa 32
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<212> DNA
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gcaagcttct accacaggac tcctgcct 28

Claims (8)

1. dengue 2-type virus NS5 PROTEIN Cs end truncated segment, it is characterised in that its amino acid sequence such as SEQ ID NO:Shown in 1, Or the sequence is through replacing, lacking or adding one or several amino acids formed amino acid sequences with equal function.
2. truncated segment recombinant polypeptide NS5/C70 preparation method described in the claim 1 containing His labels, it is characterised in that Comprise the following steps：
(1) recombinant plasmid pQE30-NS5/C70 structure：Using the plasmid containing dengue 2-type virus NS5 protein coding genes as mould Plate, designs primer, enters performing PCR amplification, obtained amplified production BamH I, Hind III double digestions reclaim small pieces after purification Section is connected with linear carrier pQE30, obtains recombinant plasmid pQE30-NS5/C70；
(2) structure of prokaryotic expression system：Recombinant plasmid pQE30-NS5/C70 is transformed into E.Coli competence M15 [pREP4] In, obtain recombinant bacterium M15/pQE30-NS5/C70；
(3) recombinant polypeptide NS5/C70 expression and purifying；
Wherein, the primer of step (1) is：
- the GG of forward primer F1 5 'GGATCCCCAAACCTAGACATAATCGGGAAA-3’
- the GC of reverse primer R1 5 'AAGCTTCTACCACAGGACTCCTGCCT-3’。
3. method according to claim 2, it is characterised in that step (3) is specially：
1. Zengjing Granule stage：Recombinant bacterium M15/pQE30-NS5/C70 is inoculated in 2 × YT culture mediums containing Amp and Kana, The glucose of addition 1%, is cultivated to OD under 180rpm, 37 DEG C of aeration conditions570For 0.7-0.8；
2. induction period：IPTG to final concentration of 1mM is added, in 25 DEG C, 180rpm inductions 4h；
3. supernatant is collected by centrifugation and with 0.45 μm of membrane filtration；Filtrate is collected with affinity chromatography to be purified, then through various concentrations Imidazole solution gradient elution, finally give the NS5/C70 recombinant polypeptides of purifying.
4. dengue 2-type virus NS5 PROTEIN Cs end truncated segment described in claim 1 is being prepared for different serotypes dengue virus Application in the immunological diagnostic reagent of infection.
5. it is used for different serotypes Dengue disease as prepared by dengue 2-type virus NS5 PROTEIN Cs end truncated segment described in claim 1 The immunological diagnostic reagent of poison infection.
6. dengue 2-type virus NS5 PROTEIN Cs end truncated segment described in claim 1 is being prepared for different serotypes dengue virus Application in the colloidal gold immunochromatographimethod diagnosis test paper of infection.
7. the colloidal gold immunochromatographimethod diagnosis test paper for dengue virus infection, it is characterised in that the test strips are by sample Pad, colloidal gold pad, NC films, absorbent filter and PVC bottom plates composition, sample pad, colloidal gold pad, NC films and absorbent filter are mutual successively Superposition is pasted onto on PVC bottom plates；
Wherein, the anti-colloidal gold composite of goat-anti people two is coated with colloidal gold pad；NC films are provided with p-wire and nature controlling line, institute State that p-wire is fixed with dengue 2-type virus NS5 PROTEIN Cs end truncated segment described in claim 1 or the restructuring containing His labels is more Peptide, the nature controlling line is fixed with SPA.
8. it is used for various dengue virus sense containing diagnosis test paper described in diagnostic reagent described in claim 5 or claim 7 The quick detection kit of dye.
CN201710322563.6A 2017-05-09 2017-05-09 Dengue 2-type virus NS5 PROTEIN Cs end truncated segment and preparation method and application Pending CN107226848A (en)

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CN108395470A (en) * 2018-01-10 2018-08-14 北京工业大学 With the small peptide and its application for inhibiting dengue virus duplication effect
CN114585750A (en) * 2019-10-03 2022-06-03 新加坡科技研究局 Method for detecting or differentiating chikungunya virus, dengue virus and Zika virus

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CN108395470A (en) * 2018-01-10 2018-08-14 北京工业大学 With the small peptide and its application for inhibiting dengue virus duplication effect
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Application publication date: 20171003