CN110066343A

CN110066343A - A kind of recombinant antigen and its application for detecting HIV new infections

Info

Publication number: CN110066343A
Application number: CN201910433891.2A
Authority: CN
Inventors: 刘兴旺; 孟婷婷
Original assignee: Beijing Xinchuang Bioengineering Co Ltd
Current assignee: Beijing Xinchuang Bioengineering Co Ltd
Priority date: 2019-05-23
Filing date: 2019-05-23
Publication date: 2019-07-30
Anticipated expiration: 2039-05-23
Also published as: CN110066343B

Abstract

The recombinant antigen and its application that the invention discloses a kind of for detecting HIV new infections, it is related to antigen preparation technical field, recombinant antigen includes B amino acid sequence, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation, hydrophilic amino acid core sequence and the lysine sequence of antigen both ends addition, B amino acid sequence is as shown in SEQ ID No.1, E amino acid sequence is as shown in SEQ ID No.2, D amino acid sequence is as shown in SEQ ID No.3, hydrophilic amino acid core sequence is as shown in SEQ ID No.4, BC series of variation is as shown in SEQ ID No.5, AE series of variation is as shown in SEQ ID No.6.The solubility of the recombinant antigen is high, is easy to purify, is easy to mark, and erroneous judgement caused by the false positive reaction and hypotype when being avoided that or reduce HIV detection new infections are omitted improves the accuracy of detection.

Description

A kind of recombinant antigen and its application for detecting HIV new infections

Technical field

The present invention relates to antigen preparation technical fields, in particular to a kind of for detecting the recombination of HIV new infections Antigen and its application.

Background technique

AIDS (Human immunodeficiency virus, HIV) seriously threatens human health, is that the whole world is common The great public health problem faced." AIDS Epidemic report " display of The Joint Programme on AIDS publication, whole world survival Patients infected hiv and patient up to 34,000,000 people, annual 2500000 people of new infections, dead 1,700,000 people, AIDS is anti- It is still severe to control situation.

Currently, the infection population of AIDS spreads through sex intercourse in diversification and has become the main path of aids transmission, simultaneously Intravenous drug use secondhand injectors, unlicensed prostitute crowd's condom utilization rate is lower and the risk factors such as male same sexual behaviour are widely present, So that Prevention & Control of AIDS work still faces huge challenge.

The detection of AIDS virus new infections is the important content of AIDS Surveillance work, for understanding HIV spread in time Dynamically, people at highest risk is found, assessment control effect is of great significance.In recent years, with the development of biotechnology, so that utilizing Laboratory method judgement is infected recently, and then estimates that disease incidence becomes research hotspot.

But the antigen research currently used for the detection of AIDS virus new infections is seldom, and false sun rate is high.

Summary of the invention

The first object of the present invention is to provide a kind of for detecting the recombinant antigen of HIV new infections, the recombinant antigen Solubility it is high, be easy to purify, can be avoided or reduces the false positive reaction occurred when the detection of HIV new infections, improve and detect Sensitivity and accuracy.

The second object of the present invention is to provide a kind of nucleic acid point of recombinant antigen for code detection HIV new infections Son can encode a kind of recombinant antigen suitable for detecting the detection of AIDS virus new infections, and the recombinant antigen has The high advantage of Detection accuracy.

The third object of the present invention is to provide a kind of expression vector comprising above-mentioned coding is for detecting AIDS virus The nucleic acid molecules of the recombinant antigen of new infections detection.

The fourth object of the present invention is to provide a kind of expression bacterial strain comprising has above-mentioned new for detecting AIDS virus The expression vector of the recombinant antigen of hair infection detection.

The fifth object of the present invention is to provide a kind of for detecting the detection kit of HIV new infections, detection examination Agent box can accurately detect AIDS virus new infections.

The present invention is implemented as follows:

The embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections comprising B amino acid sequence Column, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, it is described Hydrophilic amino acid core sequence is set between B, E, D amino acid sequence, BC and AE series of variation；

The B amino acid sequence is as shown in SEQ ID No.1, and the E amino acid sequence is as shown in SEQ ID No.2, institute D amino acid sequence is stated as shown in SEQ ID No.3, the hydrophilic amino acid core sequence is described as shown in SEQ ID No.4 BC series of variation is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.

It is above-mentioned for detecting the core of the recombinant antigen of HIV new infections for encoding that the embodiment of the invention also provides a kind of Acid molecule, the nucleic acid molecules include B nucleic acid molecules, E nucleic acid molecules, D nucleic acid molecules, BC nucleic acid molecules, AE nucleic acid molecules with And hydrophilic nucleotide sequence；

The nucleotide sequence of the B nucleic acid molecules is as shown in SEQ ID No.8, the nucleotide sequence of E nucleic acid molecules such as SEQ Shown in ID No.9, the nucleotide sequence of D nucleic acid molecules is as shown in SEQ ID No.10, hydrophilic nucleotide sequence such as SEQ ID Shown in No.11, the nucleotide sequence of the BC nucleic acid molecules is as shown in SEQ ID No.12, the nucleotide of the AE nucleic acid molecules Sequence is as shown in SEQ ID No.13.

The embodiment of the invention also provides a kind of expression vectors comprising has above-mentioned nucleic acid molecules；

Preferably, the expression vector is pET28a.

The embodiment of the invention also provides a kind of expression bacterial strains comprising has above-mentioned expression vector；

Preferably, the expression bacterial strain is E. coli expression strains；

Preferably, the expression bacterial strain is e. coli bl21 (DE3).

It is a kind of for detecting the detection kit of HIV new infections comprising above-mentioned recombinant antigen or above-mentioned nucleic acid point Son.

The invention has the following advantages:

The embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections, the Recombinant antigen B amino acid Sequence, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, parent Aqueous amino acid core sequence is set between B, E, D amino acid sequence, BC and AE series of variation；B amino acid sequence such as SEQ Shown in ID No.1, E amino acid sequence is as shown in SEQ ID No.2, and D amino acid sequence is as shown in SEQ ID No.3, hydrophily Amino acid core sequence is as shown in SEQ ID No.4, and BC series of variation is as shown in SEQ ID No.5, and the AE series of variation is such as Shown in SEQ ID No.6.The solubility of the recombinant antigen is high, is easy to purify, when can be avoided or reduce the detection of HIV new infections False positive reaction, improve the sensitivity and accuracy of detection.

It include above-mentioned for detecting the nucleic acid of the recombinant antigen of HIV new infections the embodiment of the invention also includes having a kind of Molecule, the expression vector including the nucleic acid molecules, the expression bacterial strain including the expression vector and including above-mentioned recombinant antigen or The detection kit for being used to detect HIV new infections of nucleic acid molecules, the detection kit have the advantages that detection accuracy is high.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is that the HIV-Ag11 in the embodiment of the present invention 2 expresses SDS-PAGE electrophoresis after bacterial strain ultrasonication；

Fig. 2 is HIV-Ag11 SDS-PAGE electrophoresis after purification in the embodiment of the present invention 3.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Below specifically to the recombinant antigen for being used to detect HIV new infections in the embodiment of the present invention and its using progress It is bright.

The laboratory testing method of new infections, can be there are many method according to the marker difference of detection.Serology Method before sun turns mainly has viral nucleic acid (Ribonucleic acid, RNA) detection and the detection of p24 antigen；Serology sun turns Afterwards, according to testing principle difference, be roughly divided into 6 classes: antibody titer detection, affinity of antibody detection, specific antibody Zhan always resist The ratio detection of body, is linearly immunized p24 antigen immunoglobulin 3 antibody (Immunoglobulin G3, IgG3) antibody test Trace detection, anti-specific immunity antigenicity reaction detection.Wherein, by the original of the total antibody ratios of HIV gp41 specific antibody Zhan The HIV-1 BED of reason exploitation captures EIA method (BED-CEIA), abbreviation BED method.

The mature antigen for being currently used for BED method is artificial chemically synthesized polypeptide, and specially artificial chemistry is respectively synthesized B, E, D polypeptide, then 3 polypeptides are linked together with chemical coupling method, the preparation process of the polypeptide is complicated, antigen yield is low, It is at high cost, and technique is unstable, repetition differs, difference between batch is big, brings great difficulty for the application of antigen.Gene work Then dissolubility is poor, label is difficult for the BED antigen of journey recombination, false positive rate is high, is not used to the exploitation of BED method kit.

The embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections comprising has B amino acid sequence Column, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, it is described Hydrophilic amino acid core sequence is set between B, E, D amino acid sequence, BC and AE series of variation.

B amino acid sequence is as shown in SEQ ID No.1, and the E amino acid sequence is as shown in SEQ ID No.2, the D ammonia Base acid sequence is as shown in SEQ ID No.3, and as shown in SEQ ID No.4, the BC becomes the hydrophilic amino acid core sequence Different sequence is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.The recombinant antigen has solubility Height, the false positive rate of detection HIV new infections is low, is easy to purify, and the recombinant antigen that joined AE series of variation and BC series of variation is suitable For the detection of China's HIV new infections, have the advantages that detection accuracy is high.

Further, the N-terminal and C-terminal for detecting the recombinant antigen sequence of HIV new infections are added with for improving Antigen can be markup lysine sequence.

Further, in the sequence of the recombinant antigen, the B amino acid sequence, E amino acid sequence, D amino acid sequence Column, the BC series of variation and AE series of variation are successively arranged successively from the N-terminal of recombinant antigen to C-terminal.

It is further preferred that the sequence of the recombinant antigen for detecting HIV new infections is as shown in SEQ ID No.7.

It is above-mentioned for detecting the core of the recombinant antigen of HIV new infections for encoding that the embodiment of the invention also provides a kind of Acid molecule, the nucleic acid molecules include B nucleic acid molecules, E nucleic acid molecules, D nucleic acid molecules, BC nucleic acid molecules and AE nucleic acid molecules with And hydrophilic nucleotide sequence.

The nucleotide sequence of B nucleic acid molecules is as shown in SEQ ID No.8, the nucleotide sequence of E nucleic acid molecules such as SEQ ID Shown in No.9, the nucleotide sequence of D nucleic acid molecules is as shown in SEQ ID No.10, hydrophilic nucleotide sequence such as SEQ ID No.11 Shown, the nucleotide sequence of BC nucleic acid molecules is as shown in SEQ ID No.12, the nucleotide sequence such as SEQ of the AE nucleic acid molecules Shown in ID No.13.

Further, the nucleotide sequence of nucleic acid molecules is as shown in SEQ ID No.14.

The embodiment of the invention also provides a kind of expression vectors comprising has above-mentioned nucleic acid molecules.Preferably, the expression Carrier is pET28a.

Further, the embodiment of the invention also provides a kind of expression bacterial strains comprising has above-mentioned expression vector.It is preferred that Ground, the expression bacterial strain are E. coli expression strains；Preferably, the expression bacterial strain is e. coli bl21 (DE3).

The embodiment of the invention also provides a kind of for detecting the detection kit of HIV new infections comprising above-mentioned use In the recombinant antigen of detection HIV new infections or the nucleic acid molecules of the above-mentioned recombinant antigen for detecting HIV new infections.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

It is provided in this embodiment a kind of for detecting the recombinant antigen of HIV new infections comprising B amino acid sequence, E ammonia Base acid sequence, D amino acid sequence, BC series of variation and AE series of variation, hydrophilic amino acid core sequence and recombinant antigen N The lysine sequence at end and C-terminal addition.

B amino acid sequence, E amino acid sequence, D amino acid sequence, the BC series of variation and AE series of variation successively from The N-terminal of recombinant antigen is arranged successively to C-terminal.The hydrophilic amino acid core sequence be set to B, E, D amino acid sequence and Between BC series of variation and AE series of variation.

Specifically, B amino acid sequence is as shown in SEQ ID No.1, the E amino acid sequence as shown in SEQ ID No.2, The D amino acid sequence is as shown in SEQ ID No.3, and the hydrophilic amino acid core sequence is as shown in SEQ ID No.4；BC Series of variation is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.

Further, be added with can be markup for improving antigen for the N-terminal of the amino acid sequence of recombinant antigen and C-terminal Lysine sequence.

In the present embodiment, recombinant antigen is named as HIV-Ag11, the amino acid sequence of recombinant antigen such as SEQ ID No.7 It is shown.

Embodiment 2

For detecting the building of the recombinant antigen of HIV new infections

1. target gene fragment connects transformed clone bacterium

The B/E/D-pUC plasmid (B/E/D nucleic acid molecules) and BC/AE-pUC plasmid (BC/AE nucleic acid molecules) of gene chemical synthesis Comprising recombinant antigen sequence sequence, using pET28a as expression vector, initiation codon contains His label, merges table The albumen reached is purified using Ni column.

Specifically, B/E/D-pUC plasmid carries out double digestion, BC/AE-pUC plasmid EcoR I and Xho with Nde I and EcoR I I digestion, pET28a use Nde I and I digestion of Xho as skeleton, and digestion system is carried out according to table 1,30 DEG C of digestion 30min.This experiment Restriction enzyme enzyme cutting used is purchased from Beijing Quanshijin Biotechnology Co., Ltd, is the fast enzyme cutting of its Flycut series.

1 double digestion system of table

Enzyme 1	1μl
		Enzyme 2	1μl
10×buffer	2μl
		Substrate	16μl
Total	20μl

Three digestion systems all do agarose gel electrophoresis and DNA purification and recovery, and used kit is purchased from Beijing ancient cooking vessel state Prosperous biotechnology Co., Ltd, glue recovery product are connected 16 hours by linked system shown in table 2, DNA ligase Solution I is purchased from TAKARA, converts escherichia coli DH5a, obtains connection transformed clone bacterium, and escherichia coli DH5a competence is thin Born of the same parents are purchased from Beijing Quanshijin Biotechnology Co., Ltd.

2 linked system of table

Genetic fragment B/E/D	2μl
		Genetic fragment AE/BC	2μl
pET28a	1μl
		SoutionⅠ	5μl
Total volume	10μl

2. positive spot screening and plasmid extract

The plated growth of connection transformed clone bacterium in step 1 has white Escherichia coli list spot, and picking plate list spot carries out Bacterium colony PCR, PCR primer are as follows:

pET-fw:CACTATAGGGGAATTGTGAGCGGATAAC(SEQ ID No.15)。

pET-rv:CTCAGCTTCCTTTCGGGCTTTGTTAG(SEQ ID No.16)。

Bacterium colony PCR reaction system component is shown in Table 3, PCR response procedures are as follows: initial denaturation: 94 DEG C of 5min；Circulation: 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 90s, totally 30 times circulation；Extend: 72 DEG C of 5min.Positive spot colony PCR product clip size theory is 1034bp。

3 bacterium colony PCR reaction system of table

Template DNA	0.5μl
		pET-fw(20μM)	1μl
pET-rv(20μM)	1μl
		2×TaqMix	10μl
ddH₂O	7.5μl
		Total volume	20μl

It by the single spot of the positive screened, shakes bacterium and stays overnight, next day extracts plasmid, and plasmid extraction kit is that OMEGA plasmid is small Extraction reagent kit.The HIV-Ag11 plasmid of acquisition, plasmid double digestion (I+Xho I of Nco) are accredited as the positive, raw work bioengineering are sent to have The sequencing of Beijing sequencing portion, limit company, sequence and design are completely the same, can be used to conversion Bacillus coli expression bacterium.

3.HIV-Ag11 plasmid converts Bacillus coli expression bacterium BL21 (DE3)

1 μ l of HIV-Ag11 plasmid is taken, 50 μ l e. coli bl21 (DE3) competent cells are added, place 30min on ice, 42 DEG C of heat shock 90s place 2min on ice, and 500 μ l LB culture mediums (being free of antibiotic) is added, and 37 DEG C, 200rpm is cultivated 1 hour, 60ul culture solution spread plate (containing 50 μ g/ml of kanamycin sulfate) is taken, obtains including the big of recombinant antigen HIV-Ag11 Enterobacteria expresses bacterium BL21 (DE3).E. coli bl21 (DE3) competent cell used is tested purchased from the complete biological skill of formula gold in Beijing Art Co., Ltd.

4. Escherichia coli inducing expression

The glycerol stock for expressing bacterium accesses 300ml culture medium (containing 50 μ g/ml of kanamycin sulfate), and 37 DEG C, 200rpm shakes Bottle is cultivated to OD₆₀₀=0.8, it is added IPTG inducing expression (the final concentration of 0.7mM of IPTG), continues to cultivate 4h.By bacterium solution 6500rpm is centrifuged 10min, abandons supernatant, collects bacterium mud.

5. Escherichia coli ultrasonication

10mMTris-HCl (pH8.0) is added in the ratio of 1g bacterium mud 40ml buffer in the Escherichia coli bacterium mud of expression, weight Outstanding thallus, the ultrasonication on ultrasonic machine, 20ml bacterium solution, 200w, ultrasonic 2s stop 2s ultrasound 20min.Ultrasonic complete bacterium solution compared with Be it is limpid, take 50 μ l ultrasound liquid, be centrifuged 10min precipitation and separation and supernatant through 11000rpm, precipitating is used as inclusion body sample.It takes Clear 30 μ l carries out whether having purpose protein expression in SDS-PAGE detection precipitating and supernatant, please refers to attached drawing as Supernatant samples 1, HIV-Ag11 protein expression form is inclusion body expression, and inclusion body is taken to carry out follow-up test.

Embodiment 3

For detecting the purifying of the recombinant antigen of HIV new infections.

The broken thallus that 2 step 5 of embodiment is obtained is usedJ-E Beckman centrifuge is centrifuged, from Mental and physical efforts: 22000g, temperature: 4-8 DEG C, the time: 15 minutes.Supernatant is abandoned, inclusion body precipitating is retained.

By inclusion body with by precipitating inclusion body lysate (10mM Tris-HCl+500mM NaCl pH 8.0+8mM Urea it) dissolves, after inclusion body cracks completely, 22000g, centrifugation 15min removes impurity, retains supernatant, i.e. Ni Sepharose Fast Flow column chromatography samples.

The Ni Sepharose Fast Flow gel content of suitable chromatographic column and filling is selected to carry out dress column.It will fill Ni Sepharose Fast Flow column chromatography water afterwards rinses 4 column volumes.With the equilibrium liquid (10mM of 4 times of column volumes Tris-HCl+500mM NaCl pH 8.0+8mM Urea) balance purification column, loading after the completion of balance.After end of the sample, with flat Remaining sample in the liquid flushing pipeline that weighs and chromatographic column.With removal of impurities liquid (the 10mM Tris-HCl pH 8.0+500mM of 4 times of column volumes NaCl+30mM IMZ+8mM Urea) elution removing impurity.With eluent (the 10mM Tris-HCl pH 8.0+ of 4 times of column volumes 500mM NaCl+200mM IMZ+8mM Urea) elution of bound antigen protein, with the CB of 50mM pH9.6 dialysis removal denaturation After agent and imidazoles, recombinant antigen as after purification.

This test is gone back while being constructed the antigen HIV-BED of BED polypeptide expressing in series, expression vector pET28a, expression Bacterium is BL21 (DE3), and protein expression form is also inclusion body expression.Use inclusion body lysate (10mM Tris-HCl+500mM NaCl pH 8.0+8mM Urea) dissolution, inclusion body cracking is incomplete, after taking the hydrotropies measures such as 42 DEG C of processing, ripples ultrasounds It can not still crack completely.After its lysate is centrifuged removal precipitating, there are HIV-BED albumen, use and HIV-Ag11 in supernatant Identical method (Ni column) is purified.SDS-PAGE detection is carried out after purification, and testing result please refers to attached drawing 2 and table 4.

4 HIV-Ag11 and HIV-BED inclusion body of table cracks situation comparison

By table 4 and Fig. 2 it is found that for the HIV-BED antigen of the recombinant antigen that provides of embodiment 1 compared with the existing technology, More easily cracking purification.

Embodiment 4

The specificity and Detection of Stability for the recombinant antigen for detecting HIV new infections that embodiment 3 provides.

Goat anti-human igg is coated with 96 orifice plates, enzyme combination diluent, A liquid, B liquid, terminate liquid and is purchased from Ying Kexin wound (Xiamen) Science and Technology Ltd..

Fused antigen label horseradish peroxidase (Over-voltage protection): weighing 10mgHRP and be dissolved in 1ml ultrapure water, room The lower stirring of temperature dissolves it sufficiently, and the 0.1M NaIO that 0.2ml newly matches is added in upper liquid₄Solution is protected from light stirring 25 minutes；Oxygen The ethylene glycol solution after 0.2ml dilution is added after the completion of change in upper liquid, continues to be protected from light stirring 30 minutes；Above-mentioned reaction solution is added In HIV-Ag11 protein solution after to dialysis, dialysis 4.5h is carried out with 50mM pH9.5 carbonate buffer solution after mixing, it is intermediate It changes the liquid once；The 10mg/ml NaBH that 0.4ml newly matches is added₄Solution mixes, then sets 4 DEG C of 2h；Above-mentioned liquid is packed into bag filter In, it is dialysed with 20mM TBS pH8.0 buffer, at room temperature overnight, liquid is changed twice in centre；Next day adds into obtained finished product enzyme The glycerol of equivalent mixes, -20 DEG C of preservations.

Enzyme-labelled antigen Activity determination:

Required amount of capillary strip for being coated with goat anti-human igg is taken, is placed on grillage, and carries out mark.Distinguish in order 100 μ l key quality-control products and positive and negative control are added in corresponding aperture.It sets in 37 DEG C of insulating boxs and incubates 60min, filled with cleaning solution Divide washing 5 times, then detains dry (cleaning solution soaking time 30-60 seconds).

100 μ l enzyme-labelled antigens (1:5000 dilution) is added in every hole respectively, is placed in 37 DEG C of insulating boxs and incubates 30min (can onboard cover sealing plate film if needed) is sufficiently washed 5 times with cleaning solution, and then button is dry.It is each that substrate A, B liquid is added in every hole 50 μ l after patting mixing, are placed in 37 DEG C of insulating boxs and incubate 30min.50 μ l of terminate liquid is added in every hole, mixes.With microplate reader list Wavelength 450nm or dual wavelength 450/630nm measure each hole A value (when being measured with Single wavelength, need to use blank control air-conditioning zero, one As use dual wavelength method), and record result.

UtoffValue (COV) is calculated: (negative control OD value is lower than 0.075 for COV=negative control mean OD value × 2.0 Make 0.075 to calculate, be higher than 0.075 and calculated by practical OD value), sample to be tested OD value >=COV is the positive, sample to be tested OD value < COV is feminine gender.

Specific detection:

The HIV positive serum of confirmation 112, negative serum 1000 are detected by enzyme-labelled antigen detection method.

The HIV-Ag11 that embodiment 3 provides also carries out horseradish peroxidase-labeled using identical method, and detects work Property and specificity.Testing result please refers to table 5 and table 6.

5 enzyme-labelled antigen Activity determination result of table

6 specific detection result of table

By table 5 and table 6 it is found that the antigen active and specificity of the recombinant antigen HIV-Ag11 that embodiment 3 provides are superior to The HIV-BED of the prior art.

Accuracy detection:

The AIDS new infections serum 12 confirmed through CDC is selected, previous infection serum 10 are pressed kit test method It is detected, the HIV-Ag11 of HIV-BED and the HRP label of HRP label is selected to carry out Parallel testing.Accuracy testing result is asked Referring to table 7.

The accuracy testing result of 7 two kinds of antigens of table

As shown in Table 7, the accuracy rate of HIV-Ag11 new infections accuracy rate ratio HIV-BED is high, HIV-Ag11 detection Previous infection coincidence rate ratio HIV-BED high.

Detection of Stability:

Purified detected material HIV-Ag11 is divided into 2 parts, 1 part is placed in 4 DEG C of refrigerators, and another 1 part is placed in 37 DEG C It takes out to be put into 4 DEG C of refrigerators after 6 days in insulating box and balance overnight, at detection plate, the ELISA Plate placed with 4 DEG C is closed coating with positive Key quality-control product carries out Detection of Stability.Detection of Stability result please refers to table 8.

Remarks: coating reaction plate is to the detection A value of positive crucial quality-control product with 4 DEG C of ELISA Plates to positive crucial quality-control product The ratio of detection A value is judged that ratio more high stability is better.

8 Detection of Stability result of table

As shown in Table 8, the stability for the recombinant antigen HIV-Ag11 that embodiment 3 provides is preferable.

To sum up, the embodiment of the invention provides a kind of for detecting the recombinant antigen of HIV new infections, the Recombinant antigen B Amino acid sequence, E amino acid sequence, D amino acid sequence, BC series of variation, AE series of variation, hydrophilic amino acid core sequence And the lysine sequence of recombinant antigen both ends addition, hydrophilic amino acid core sequence are set to B, E, D amino acid sequence, BC And between AE series of variation；B amino acid sequence as shown in SEQ ID No.1, E amino acid sequence as shown in SEQ ID No.2, D amino acid sequence is as shown in SEQ ID No.3, and hydrophilic amino acid core sequence is as shown in SEQ ID No.4, BC series of variation As shown in SEQ ID No.5, the AE series of variation is as shown in SEQ ID No.6.The solubility of the recombinant antigen is high, is easy to mention It is pure, it is easy to mark, can be avoided or reduce the false positive reaction when detection of HIV new infections, improve the sensitivity and standard of detection True property.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

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<213>artificial sequence

<400> 10

ctgcaggcgc gcattctggc gattgaacgc tatctgcagg atcagcagct gctgggcatt 60

tggggctgca gcggcaaaca tatttgcacc accacc 96

<210> 11

<211> 57

<212> DNA

<213>artificial sequence

<400> 11

gataaagata gcacccagca gaacaccggc ggcccgcaga ccaccagcaa aggcggc 57

<210> 12

<211> 102

<212> DNA

<213>artificial sequence

<400> 12

ctccaaaccc gtgttctcgc gattgaacgt tacctgaaag accagcaact gctcggtatc 60

tggggttgct ctggcaaact catctgcact accgcagttc cg 102

<210> 13

<211> 102

<212> DNA

<213>artificial sequence

<400> 13

ctccaggcgc gtgtgctggc tgtcgagcgc tacctgcagg atcagaaatt cctcggtctc 60

tggggctgct ccggcaagat tatttgtacc actgcagcac ca 102

<210> 14

<211> 894

<212> DNA

<213>artificial sequence

<400> 14

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgggcaaaa aaggcaaagg cagcctgcag gcgcgcattc tggcggtgga acgctatctg 120

aaagatcagc agctgctggg catttggggc tgcagcggca aactgatttg caccaccacc 180

gcgccgcgcg gcgataaaga tagcacccag cagaacaccg gcggcccgca gaccaccagc 240

aaaggcggca aagaatttct gcaggcgcgc gtgctggcgg tggaacgcta tctgaaagat 300

cagaaatttc tgggcctgtg gggctgcagc ggcaaaatta tttgcaccac cgcggcgccg 360

cgcggcgata aagatagcac ccagcagaac accggcggcc cgcagaccac cagcaaaggc 420

ggcgtggatc tgcaggcgcg cattctggcg attgaacgct atctgcagga tcagcagctg 480

ctgggcattt ggggctgcag cggcaaacat atttgcacca ccacccgcgg cgataaagat 540

agcacccagc agaacaccgg cggcccgcag accaccagca aaggcggcga attcctccaa 600

acccgtgttc tcgcgattga acgttacctg cgtgaccagc aactgctcgg tatctggggt 660

tgctctggcc gtctcatctg cactaccgca gttccgcgcg gcgataaaga tagcacccag 720

cagaacaccg gcggcccgca gaccaccagc aaaggcggcg tggatctcca ggcgcgtgtg 780

ctggctgtcg agcgctacct gaaggatcag cgtttcctcg gtctctgggg ctgctccggc 840

aagactattt gtaccactgc agcaccagaa tttggcggca aaaaaggcaa ataa 894

<210> 15

<211> 28

<212> DNA

<213>artificial sequence

<400> 15

cactataggg gaattgtgag cggataac 28

<210> 16

<211> 26

<212> DNA

<213>artificial sequence

<400> 16

ctcagcttcc tttcgggctt tgttag 26

Claims

1. a kind of for detecting the recombinant antigen of HIV new infections, which is characterized in that it includes B amino acid sequence, E amino acid Sequence, D amino acid sequence, BC series of variation, AE series of variation and hydrophilic amino acid core sequence, the hydrophilic amino Sour core sequence is set between B, E, D amino acid sequence, BC and AE series of variation；

The B amino acid sequence is as shown in SEQ ID No.1, and the E amino acid sequence is as shown in SEQ ID No.2, the D ammonia Base acid sequence is as shown in SEQ ID No.3, and as shown in SEQ ID No.4, the BC becomes the hydrophilic amino acid core sequence Different sequence is as shown in SEQ ID No.5, and the AE series of variation is as shown in SEQ ID No.6.

2. according to claim 1 for detecting the recombinant antigen of HIV new infections, which is characterized in that described for examining The N-terminal and C-terminal for surveying the recombinant antigen sequence of HIV new infections are added with lysine sequence that can be markup for improving antigen Column.

3. according to claim 1 for detecting the recombinant antigen of HIV new infections, which is characterized in that in the recombination In the sequence of antigen, the B amino acid sequence, E amino acid sequence, D amino acid sequence, the BC series of variation and AE variation sequence Leie is arranged successively from the N-terminal of recombinant antigen to C-terminal.

4. according to claim 2 for detecting the recombinant antigen of HIV new infections, which is characterized in that the recombination is anti- Former sequence is as shown in SEQ ID No.7.

5. a kind of described in any item for detecting the nucleic acid of the recombinant antigen of HIV new infections for encoding Claims 1 to 4 Molecule, which is characterized in that the nucleic acid molecules include B nucleic acid molecules, E nucleic acid molecules, D nucleic acid molecules, BC nucleic acid molecules, AE Nucleic acid molecules and hydrophilic nucleotide sequence；

The nucleotide sequence of the B nucleic acid molecules is as shown in SEQ ID No.8, the nucleotide sequence of E nucleic acid molecules such as SEQ ID Shown in No.9, the nucleotide sequence of D nucleic acid molecules is as shown in SEQ ID No.10, hydrophilic nucleotide sequence such as SEQ ID No.11 Shown, the nucleotide sequence of the BC nucleic acid molecules is as shown in SEQ ID No.12, the nucleotide sequence of the AE nucleic acid molecules As shown in SEQ ID No.13.

6. according to claim 5 for detecting the nucleic acid molecules of the recombinant antigen of HIV new infections, which is characterized in that The nucleotide sequence of the nucleic acid molecules is as shown in SEQ ID No.14.

7. a kind of expression vector, which is characterized in that it includes nucleic acid molecules described in claim 5 or 6；

Preferably, the expression vector is pET28a.

8. a kind of expression bacterial strain, which is characterized in that it contains expression vector as claimed in claim 7；

Preferably, the expression bacterial strain is E. coli expression strains.

9. expression bacterial strain according to claim 8, which is characterized in that the expression bacterial strain is e. coli bl21 (DE3).

10. a kind of for detecting the detection kit of HIV new infections, which is characterized in that it includes that Claims 1 to 4 is any Recombinant antigen or described in claim 5 or 6 for detecting the nucleic acid molecules of the recombinant antigen of HIV new infections described in.