CN111704657B

CN111704657B - HIV-2 recombinant antigen and preparation method and application thereof

Info

Publication number: CN111704657B
Application number: CN202010550127.6A
Authority: CN
Inventors: 曾红; 蔡泉蜀; 干盈盈; 何涛
Original assignee: Sichuan Ankerei New Material Technology Co ltd
Current assignee: Sichuan Ankerei New Material Technology Co ltd
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2021-11-09
Anticipated expiration: 2040-06-16
Also published as: CN111704657A

Abstract

The invention relates to an HIV-2 recombinant antigen, the amino acid sequence of which is shown as SEQ ID NO.1 or a derivative amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the amino acid sequence shown as SEQ ID NO.1, wherein the derivative amino acid sequence has the activity of the amino acid sequence shown as SEQ ID NO. 1. The recombinant proteins of the invention are useful for HIV detection and have improved performance in developing in vitro diagnostic kits. The invention also relates to corresponding polynucleotides, constructs, host cells, fusion proteins, compositions, kits and methods of preparation.

Description

HIV-2 recombinant antigen and preparation method and application thereof

Technical Field

The invention relates to the field of HIV detection, in particular to a recombinant antigen for HIV-2 immunodetection.

Background

AIDS (acquired immune deficiency syndrome, AIDS) is a viral disease which seriously harms human health and is in the spotlight in the world today. The major etiological agents responsible for AIDS are the human immunodeficiency viruses type I and type II (HIV-1 and HIV-2). HIV-infected individuals die as a result of the progression of immunodeficiency, from asymptomatic carriers to severe immune failure, leading to the development of a variety of fatal diseases. According to WHO statistics, the number of global HIV-infected people is estimated to be 3790 ten thousand by 2018, and 170 thousand of global new HIV-infected people are only 2018. In developing countries, due to various reasons such as economy, culture and education, the prevalence of HIV is getting more and more intense, and some countries have lost control over the prevalence of diseases, while our country tends to be in the early stage of explosive prevalence.

The HIV gene has a total length of about 9.8kb, and contains 3 structural genes (gag, pol and env), 2 regulatory genes (tat transactivator and rev virion protein expression regulator) and 4 auxiliary genes (nef negative regulatory factor, vpr virus r protein, vpu virus u protein and vif virion infectious factor). HIV is a virus with strong variability, the degree of variation of each gene is different, and the env gene variation rate is the highest.

Based on the difference of HIV genes, the genes are classified into HIV-1 type and HIV-2 type. Between HIV-1 and HIV-2, the nucleotide sequence has 40-60% homology.

Based on the homology of the env gene coding for the envelope protein and the gag gene sequence coding for the capsid protein, HIV-1 types were further divided into 3 groups, the M subtype group (main, i.e., main group), the O subtype group (outline, i.e., peripheral group) and the N subtype group (new, non-M, non-O new or non-M non-O group), wherein the M group had A, B, C, D, E, F, G, H, I, J, K11 subtypes. In addition, in recent years, a number of prevalent recombinants have been discovered. Group O was isolated from karyon and galbanum in 1990 and had only 50% homology to the amino acid sequences of the other subtypes of group M. The N groups were recently isolated from two patients with karilon, and on the phylogenetic tree, they were called N groups because they were a group of new viruses that did not belong to either the M or O group.

The biological properties of HIV-2 are similar to those of HIV-1, and there are at least A, B, C, D, E, F, G7 subtypes.

China takes HIV-1 as a main epidemic strain. However, few HIV-2 infected people were discovered and confirmed in some areas since 1999. The method for timely discovering HIV infectors and identifying HIV subtypes has important significance for tracking epidemic trend, timely making diagnosis, developing new diagnostic reagents, developing new drugs and developing vaccines.

The prevention and treatment direction of AIDS mainly comprises three aspects of vaccine, diagnosis and treatment, wherein the development of HIV vaccine is not successful until now; HIV treatment is not only expensive, but also extremely limited in efficacy; this makes the diagnosis of HIV the most basic and effective means for aids prevention.

Since the first generation ELISA reagent appeared in 1985, with the rapid development of medical technology, the coating antigen has been developed from the first generation whole virus lysate to the third generation double-antigen sandwich reagent which has been subjected to gene recombination, polypeptide antigen coating and labeling and has good sensitivity and specificity, the detection subtypes comprise HIV-1, HIV-2 and HIV-1 type O subtypes, and the window period is shortened from 10 weeks to 3-4 weeks. To avoid the window-phase infection, fourth generation ELISA screening reagents have been developed that simultaneously detect antigen and antibody.

The quality of the antigen is the most critical factor in determining the performance of the HIV detection reagent. The improvement of HIV detection performance lies in the sensitivity and specificity of detection so as to ensure the accuracy of detection results, and also lies in the stability of reagents so as to realize detection under a more complex environment and obtain accurate results.

Accordingly, there is a strong need in the art for improved HIV antigens to improve the performance of HIV detection reagents.

Disclosure of Invention

In order to improve the performance of the HIV detection reagent, the inventor researches the truncated HIV-2gp36 antigen, and selects an HIV-2 recombinant gp36 antigen which has high specificity and good stability and meets the development requirement of an immunodiagnostic reagent from a plurality of different truncated antigens through a large number of experiments, thereby completing the invention.

In a first aspect, the present invention provides an HIV-2 recombinant antigen, said recombinant antigen:

1) comprises an amino acid sequence shown as SEQ ID NO. 1; or

2) A derivative amino acid sequence obtained by substituting, deleting or adding one or several (e.g., two, three, four, five or six) amino acids from the amino acid sequence in 1), wherein the derivative amino acid sequence has the activity of the amino acid sequence shown in 1).

It is noted that the recombinant antigen of the present invention is a truncated form of the recombinant antigen of HIV-2gp 36. As demonstrated in the examples section below, the recombinant antigens of the invention produced kits that tested higher rates of compliance (100%) with existing kits when compared to other structurally similar truncated gp36 recombinant antigens.

In a second aspect, the present invention provides a polynucleotide sequence which:

a) an amino acid sequence encoding a recombinant antigen of the invention; or

b) Complementary to a).

In a specific embodiment, the polynucleotide sequence of the invention is as shown in SEQ ID NO. 5.

In a third aspect, the present invention provides a polynucleotide construct comprising a polynucleotide of the invention.

In a fourth aspect, the invention provides a host cell comprising a polynucleotide construct of the invention or a polynucleotide of the invention.

In a fifth aspect, the invention provides a fusion protein comprising a recombinant antigen of the invention, and one or more additional proteins.

In some embodiments, one or more additional proteins may be other antigens for the detection of HIV antibodies.

In particular embodiments, the additional antigen for detecting HIV antibodies may be the HIV-2gp41 antigen and/or the HIV-2gp120 antigen.

In some embodiments, adjacent recombinant antigens and additional proteins, and adjacent additional proteins, may be linked by linkers.

In a sixth aspect, the present invention provides a composition for detecting HIV comprising a recombinant antigen of the invention or a fusion protein of the invention.

In some embodiments, the recombinant antigens of the invention can be coated on a solid support.

In some embodiments, the fusion protein of the invention can be coated on a solid support.

In some embodiments, the recombinant antigens of the invention may carry a detectable label.

In some embodiments, the fusion protein of the invention may carry a detectable label.

In a seventh aspect, there is provided the use of a recombinant antigen or fusion protein of the invention in the manufacture of a kit for the detection of HIV antibodies.

In some embodiments, the kits of the invention are used to detect HIV-1 antibodies and HIV-2 antibodies.

It will be appreciated that in the case of detection of HIV-1 and HIV-2, the kit of the invention may additionally comprise reagents for the detection of HIV-1.

In some embodiments, the kits of the invention are used to detect HIV-2 antibodies.

In an eighth aspect, the present invention provides a method for preparing an HIV-2 recombinant antigen, comprising:

1) introducing a polynucleotide encoding a recombinant antigen of the invention into a host cell, or transferring a polynucleotide construct comprising the polynucleotide into a host cell;

2) culturing the host cell of step 1) under conditions suitable for the production of a recombinant antigen; and

3) optionally isolating the recombinant antigen from the culture obtained in step 2).

In a ninth aspect, the present invention provides a recombinant antigen produced by the production method of the present invention.

The invention obtains the following beneficial effects:

on one hand, by optimizing the interception position of HIV-2gp36 antigen, the invention improves the specificity of HIV detection to the maximum extent.

On the other hand, the sensitivity of the recombinant antigen of the invention is proved to meet the development requirement of diagnostic reagents through national reference substance detection.

In another aspect, the recombinant antigen of the present invention has excellent thermostability, is suitable for a complicated environment while extending the storage period; in addition, the recombinant antigen has good freeze-thaw stability, and increases the frequency of repeated use. It will be appreciated that due to these properties, the cost of detection and use limitations of HIV are greatly reduced.

Drawings

FIG. 1 shows the results of identification of gp36-1 recombinant antigen induced expression in example 2. From left to right, the following are in sequence: protein Marker, gp36-1 pre-induced bacteria sample, gp36-1 post-induced bacteria sample.

FIG. 2 shows the results of identification of gp36-2 recombinant antigen induced expression in example 2. From left to right, the following are in sequence: protein Marker, gp36-2 pre-induced bacteria sample, gp36-2 post-induced bacteria sample.

FIG. 3 shows the results of identification of gp36-3 recombinant antigen induced expression in example 2. From left to right, the following are in sequence: protein Marker, gp36-3 pre-induced bacteria sample, gp36-3 post-induced bacteria sample.

FIG. 4 shows the results of identification of gp36-4 recombinant antigen induced expression in example 2. From left to right, the following are in sequence: protein Marker, gp36-4 pre-induced bacteria sample, gp36-4 post-induced bacteria sample.

FIG. 5 shows the electrophoretogram of gp36-1 recombinant antigen in example 3. From left to right, the following are in sequence: protein Marker and target protein gp 36-1.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

As mentioned above, the quality of antigen is the most critical factor affecting HIV immunoassay, so there is a need in the art for improved antigen performance to better enhance and meet the HIV in vitro detection capability. Meanwhile, because the antigen of the viral infectious disease is often large, the expression of the complete antigen protein is difficult to realize in vitro, or even if the expression in vitro is realized, the antigen protein with a large molecular weight is often not correctly folded, so that the subsequent renaturation is difficult, or the activity of the antigen obtained by renaturation is poor, thereby restricting the development of the detection reagent.

To this end, the present invention provides a novel HIV-2gp36 recombinant antigen, which selects the 553-668aa region of the HIV-2 antigen in its full length. Such recombinant antigens not only cover the dominant epitope region of gp36 antigen, but also have detection capabilities superior to other antigens slightly different from the selected region, while having superior thermostability, higher freeze-thaw stability, and immunoreactivity and sensitivity to HIV-2 type antibodies from blood.

Recombinant antigens derived in the present invention may have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to the amino acid sequence set forth in SEQ ID No. 1.

The percent identity between amino acid (or nucleic acid) sequences can be determined using standard methods known to those skilled in the art. For example, to determine the percent homology between two amino acid sequences, the sequences are aligned for optimal comparison purposes. The amino acid residues at the corresponding amino acid positions are then compared. Gaps may be introduced in one or both amino acid sequences for optimal alignment, and non-homologous sequences may be omitted for comparison purposes. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the two sequences are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences, taking into account the number of gaps introduced for optimal alignment and the length of each gap. Sequence comparisons and percent identity and similarity between two sequences can be determined using mathematical algorithms.

The kit of the present invention may take various forms well known to those skilled in the art, for example, a test strip, a test cartridge containing various reagents required for the test, a microfluidic chip, etc., and the kit may be manufactured according to known standard procedures.

In a specific embodiment, the kit of the present invention may be a colloidal gold test strip.

Kits of the invention may include containers, chips, instructions for use, buffers, immunological aids, and/or other materials, structures, and/or reagents as desired for performing the diagnosis/assay.

In some embodiments, the recombinant antigen of the present invention, when used in the preparation of a kit or composition, may be present in a container, for example, in dissolved or dried form, coated on a solid support (e.g., a membrane (e.g., NC membrane), a plate, beads (e.g., magnetic beads), microspheres (e.g., carboxyl microspheres, amino microspheres, chloromethyl microspheres, physisorption microspheres, etc.), present in a chamber of a chip in dissolved or dried form, although the present invention is not limited thereto.

Other reagents required for performing the diagnosis/detection in the kit of the present invention include, but are not limited to, other HIV antigens in addition to the recombinant antigen of the present invention, and the like. The other reactants mentioned above may be present in a manner conventional in the art, for example, in dissolved or dried form in a container, coated on a solid support, in dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.

Other materials required for performing an assay in a kit of the invention include, but are not limited to, materials for sampling, materials for performing controls, and/or materials for observing the course or result of the assay.

Other reagents required for performing the assay in the kits of the invention include, but are not limited to, buffers, detergents, visualization reagents and/or terminators.

In some embodiments, a recombinant antigen of the invention is detectably labeled when used to prepare a kit or composition. Any label and labeling method known to those skilled in the art may be used. Commonly used labels may include enzymes (e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, etc.), radioisotopes (e.g., horseradish peroxidase, beta-galactosidase, etc.), and the like³²P or¹²⁵I) Etc., biotin, digoxin, colloidal metals (e.g., colloidal gold, etc.), fluorescent dyes (e.g., fluorescein, rhodamine, Texas Red, etc.), chemiluminescent compounds or bioluminescent compounds(e.g., dioxetane, luminol, acridinium, or the like). Any labeling step well known in the art may be used, such as covalent coupling of an enzyme or biotin group, iodination, phosphorylation, biotinylation, and the like.

In some embodiments, one or more of the other reactants required for conducting the assay may also be detectably labeled.

In the kits of the invention, the recombinant antigen may be present at a suitable concentration, and one skilled in the art will be able to determine such a range depending on the particular type of kit, e.g., by reference to the concentration of gp36 antigen in a commercially available kit or by routine experimentation.

The invention includes a method of detecting the presence of HIV in a subject.

For example, the detection method includes: contacting a sample from the subject with a solid support coated with at least a recombinant antigen of the invention under conditions and for a time sufficient for the coating on the solid support to form a complex with the test agent in the sample; simultaneously or subsequently, adding a detectably labeled marker component capable of binding to the test agent to form a solid phase coating-test agent-marker component complex; subsequently, the detection result is obtained by measuring the detectable label.

For example, the detection method includes: contacting a sample from the subject with a solid phase component coated on a solid support under conditions and for a time sufficient for the solid phase component on the solid support to form a complex with the test agent in the sample; simultaneously or subsequently, adding a detectably labeled marker component comprising at least the recombinant antigen of the invention, the marker component being capable of binding to the test substance to form a solid phase component-test substance-marker component complex; subsequently, the detection result is obtained by measuring the detectable label.

Optionally, a washing step is also included after the formation of the complex to remove unbound components.

In the present invention, the sample may be selected from the group consisting of serum, plasma and blood.

Detection methods include, but are not limited to, ELISA (enzyme-linked immunosorbent assay), autoradiography, turbidimetry, fluorescence microscopy, direct and indirect enzymatic reactions, radioisotopic or nonradioisotopic methods, and the like. These methods include, inter alia, immunoturbidimetry, latex-enhanced transmission turbidimetry, Western blotting, overlay assays, RIA (radioimmunoassay) and IRMA (immunoradioimmunoassay), GIA (colloidal gold immunoassay), EIA (enzyme immunoassay), FIA (fluorescence immunoassay), and CLIA (chemiluminescent immunoassay).

The invention also relates to polynucleotides encoding recombinant antigens, e.g. for the production of fusion proteins of the recombinant antigens of the invention. Polynucleotides encoding the recombinant antigens of the invention include, but are not limited to: the coding sequence of the recombinant antigen itself; a coding sequence for a recombinant antigen and additional coding sequences; the coding sequence (and optional additional coding sequences) and non-coding sequences of the recombinant antigen may additionally include, for example, but are not necessarily limited to, one or more regulatory nucleic acid sequences, which may be regulated or regulatable promoters, enhancers, other transcriptional regulatory sequences, repressor binding sequences, translational regulatory sequences, or any other regulatory nucleic acid sequence.

Equivalent polynucleotide constructs are also encompassed by the present invention. It will be appreciated by those skilled in the art that polynucleotide constructs comprising a nucleic acid molecule encoding a recombinant antigen operably linked to an expression control sequence, and additionally comprising genetic elements such as an origin of replication and/or a selectable marker gene for maintenance and propagation in the respective host cell, are useful for the preparation of recombinant antigens by recombinant expression.

The polynucleotide construct of the invention may be, for example, a phage, plasmid or cosmid vector expressed in a prokaryotic host cell such as a bacterium (e.g., E.coli, Bacillus subtilis or Listeria); vectors for expression in yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g., Sf9 cells); viral and plasmid vectors for expression in plant cell systems (e.g., Ti plasmid, cauliflower mosaic virus CaMV; tobacco mosaic virus TMV); and viral and plasmid vectors for expression in higher eukaryotic cells or organisms. In general, such vectors are commercially available, or are available from depositories, or are available according to the teachings of publications describing the sequences, structures and methods of production of these vectors, thereby enabling their use by those skilled in the art.

In the present invention, the individual proteins in the fusion protein are connected to each other by linkers, thereby ensuring that their respective structures and activities are not affected. Exemplary linkers may be glycine-bearing flexible linkers, such as GSG, GSGGSG, GSGGSGG, GSGGSGGG, GGGGSGGG, ggggggs, SGG, and the like.

EXAMPLE 1 design of recombinant gp36 antigen and construction of expression vector

By utilizing genetic engineering technology and taking the dominant epitope region as the direction of antigen research, the inventor designs a large number of HIV-2gp36 antigen fragments, and the process of experimental screening is illustrated by taking gp36-1(SEQ ID NO.1), gp36-2(SEQ ID NO.2), gp36-3(SEQ ID NO.3) and gp36-4(SEQ ID NO.4) as examples.

Selecting a 553-668aa region of the HIV-2 antigen full length, named as gp36-1, and obtaining a nucleotide sequence gp36-1(SEQ ID NO.5) by adopting a chemical synthesis method after optimizing codons; selecting a 538-668aa region of the full length of the HIV-2 antigen, named as gp36-2, and optimizing codons to obtain a nucleotide sequence gp36-2(SEQ ID NO.6) by adopting a chemical synthesis method; selecting a 523-668aa region of the full length of the HIV-2 antigen, and naming the region as gp36-3, and obtaining a nucleotide sequence gp36-3(SEQ ID NO.7) by adopting a chemical synthesis method after optimizing codons; and selecting a 520-679aa region of the HIV-2 antigen full length, and naming the region as gp36-4, and obtaining a nucleotide sequence gp36-4(SEQ ID NO.8) by adopting a chemical synthesis method after codon optimization.

The target gene fragments gp36-1, gp36-2, gp36-3 and gp36-4 and the expression vector pTrcHisA are subjected to double enzyme digestion treatment by restriction enzymes XhoI and EcoRI respectively.

The enzyme is cut in water bath at 37 ℃ for 3h, and the cut products are detected by 1 percent TAE agarose gel electrophoresis. Recovered with QIAGEN miniprep gel recovery kit. And (3) connecting for 4h at 16 ℃, transferring the connection product into competent E.coli DH5 alpha cells by a heat shock method, coating the competent E.coli DH5 alpha cells on an LB agar culture medium containing 100 mu g/mL Amp +, and carrying out inverted culture overnight or culture for 12-14 h at 37 ℃ until a single colony grows out.

And (3) carrying out primary screening by using PCR of bacterial liquid. Specifically, the method comprises the following steps: firstly, a single colony is picked up by a sterile toothpick and put into a 1.5mL Eppendorf tube containing 1mL LB (containing 100 mu g/mL Amp +), and after shaking culture at 37 ℃ for 4h, a bacterial liquid is taken out for standby. And (3) PCR identification of bacterial liquid: after the reaction system was added, 1. mu.L of the bacterial solution was aspirated with a small gun head, mixed in the mixture, and placed in a PCR instrument to start amplification. The PCR reaction conditions are as follows: 95 ℃ for 5 min; 95 ℃, 30s, 56 ℃, 30s, 72 ℃, 1min (30 cycles of amplification); 72 ℃ for 10 min. The amplification products were detected by 1% TAE agarose gel electrophoresis.

Further characterization was performed by double-restriction enzyme characterization. Specifically, the method comprises the following steps: 10. mu.L of the clone identified as correct by PCR was aspirated into a 15mL tube containing 5mL LB (100. mu.g/mL Amp +) and cultured overnight at 37 ℃ with shaking. Plasmid extraction was performed using an Axygen miniprep plasmid kit. The restriction enzyme XhoI and EcoRI were used for double restriction. The enzyme was digested in 37 ℃ water bath for 2 h. The cleavage products were detected by electrophoresis on a 1% TAE agarose gel.

The bacterial liquid which is identified to be correct by the PCR and the enzyme digestion of the bacterial liquid is stored and then sent to the company Limited in the biological engineering (Shanghai) to be sequenced. The sequencing result is consistent with the expectation, and no base substitution, deletion or addition occurs, which indicates that the target gene is successfully cloned to the expression vector pTrcHisA and the target gene is named as pTrc-gp36-1, pTrc-gp36-2, pTrc-gp36-3 and pTrc-gp 36-4.

EXAMPLE 2 inducible expression and purification of recombinant gp36 antigen

And (3) transforming the positive recombinant plasmid subjected to enzyme digestion identification and correct sequencing into an escherichia coli E.coli BL21(Rossett) competent cell by a heat shock method, coating the competent cell on an LB agar culture medium containing 100 mu g/mL Amp + and 25 mu g/mL Chl +, and carrying out inverted culture at 37 ℃ for overnight or culture for 12-14 h until a single colony grows out.

2 single colonies were picked and inoculated into 5mL LB liquid medium containing 100. mu.g/mL Amp + and 25. mu.g/mL Chl +, and incubated at 37 ℃ and 200rpm overnight. The next day, 200. mu.L of the inoculum was transferred to 5mL of fresh LB broth containing 100. mu.g/mL Amp + and 25. mu.g/mL Chl +, and cultured at 37 ℃ and 200rpm to an OD600 of about 0.8. Taking 1mL of bacterial liquid as a sample before induction, adding IPTG to the rest of bacterial liquid until the final concentration is 1mM, and continuing to culture for 4-5 h at 37 ℃ by shaking. SDS-PAGE was performed after the induction was completed (see FIGS. 1 to 4). The strain of the clone strain liquid induced with the target protein is preserved and named as R pTrc-gp36-1, R pTrc-gp36-2, R pTrc-gp36-3 and R pTrc-gp 36-4.

The desired protein-inducing clone was selected and cultured in a large amount of 2000mL under the induction conditions determined above. The cells were collected and washed twice with 20mM PBS pH7.4 buffer. The cells were frozen at-20 ℃.

Resuspending the fermented thallus with bacteria-breaking buffer (20mM PB, 0.5M NaCl, pH 7.4), and ultrasonically breaking under ice water bath condition (ultrasonic breaking condition: power is 500W, ultrasonic is 5s, interval is 9s, and ultrasonic time is 60 min); centrifuging at 12000rpm at 10 deg.C for 20min after the ultrasound is finished; removing supernatant and collecting precipitate. The precipitate was dissolved with 20mM PB, 0.5M NaCl, 8M urea, pH7.4, stirred with a glass rod to mix well, and then dissolved with sonication (300 and 400W power, sonication 5S, interval 9S, sonication 20-30min), centrifuged at 12000rpm for 20min, the supernatant was collected and filtered through a 0.22 μ M filter.

The loading buffer solution is 20mM PB, 0.5M NaCl, 8M urea, pH7.4; the impurity washing buffer solution is as follows: 20mM PB, 0.5M NaCl, 8M Urea, 30mM imidazole, pH 7.4; the elution buffer was: 20mM PB, 0.5M NaCl, 8M urea, 500mM imidazole, pH 7.4.

Example 3 protein renaturation of recombinant gp36 antigen

Collecting the eluent, filling the eluent into a dialysis bag with the molecular weight of 3000-3500Da, and renaturing the target protein at the temperature of 2-8 ℃:

BufferA: 10mM CB, 0.05% SDS, 10% glycerol, 1mM DTT, 4M urea, pH 10.2;

BufferB：10mM CB，0.05％SDS，pH10.2。

firstly, the target protein is dialyzed by using BufferA (1L), after about 2-4 h, BufferB (2L) with the same volume is added into the BufferA, and then the target protein is dialyzed for about 2-4 h. And then, pouring 1L of dialysate containing 2M urea, adding 1L of buffer B into the dialysate, and continuing to dialyze for 2-4 h. And finally, replacing the new 2L buffer B for dialysis, and dialyzing for 3 times, wherein each time of dialysis is 2-4 h. After dialysis, the proteins were removed from the dialysis bag.

The renatured target protein is concentrated by an ultrafiltration centrifugal tube, then is centrifuged at 12000rpm at 2-8 ℃ for 20min, and the supernatant is collected in a 15mL centrifugal tube, and meanwhile, the antigen protein concentration is determined by a BCA protein quantification kit (pierce).

A small sample of the renatured protein of interest was subjected to SDS-PAGE (see FIG. 5 for the results of the electrophoresis). The size of the target protein is consistent with the expected size, and the purity of the protein is more than 85%.

Example 4 specificity of recombinant gp36 antigen

The gp36-1, gp36-2, gp36-3 and gp36-4 antigens prepared in example 3 of the invention are respectively coated on an NC membrane together with an HIV-I antigen (from Ficron, Bio-Ltd.), and colloidal gold is labeled with an HIV-I + II antigen (from Ficron, Bio-Ltd.) to prepare an antibody detection kit for human immunodeficiency virus (HIV1+ 2).

First, 100 positive samples and 500 negative samples confirmed by diagnosis were tested using HIV-1 and HIV-2 joint detection colloidal gold kits prepared according to a conventional method using gp36-2, gp36-3, gp36-4, and gp36-1 antigens, respectively, to evaluate the specificity of the antigen of the present invention. The method comprises the following specific steps: inserting a sample pad at one end of the test strip into a container containing a sample, taking out and horizontally placing after obvious sample loading is realized for about 10 seconds; the mixture was then left at room temperature and the results were observed over a period of 15 to 20 minutes. Meanwhile, the 100 positive samples and 500 negative samples were retested using a reference reagent (Mike biosystems name: human immunodeficiency virus (HIV1+2) antibody detection kit (colloidal gold method), lot number: 0917041) according to the method described in the product description, and the coincidence rate of the two test results was calculated, and the results are shown in tables 1 to 3.

TABLE 1 clinical specimen testing of gp36-2 antigen

TABLE 2 clinical specimen testing for gp36-3 antigen

TABLE 3 clinical specimen testing of gp36-4 antigen

As can be seen from tables 1-3, the antibody detection kit prepared from the gp36-2, gp36-3 and gp36-4 antigens has poor specificity and false positive results.

Next, 1580 clinical specimens, 107 HIV-type I positive specimens, 21 HIV-type II positive specimens, and 1452 negative specimens were examined according to the above-described method using an antibody detection kit prepared from pTrc-gp36-1 antigen and a reference reagent, respectively, and the results are shown in Table 4.

TABLE 4 clinical specimen testing of gp36-1 antigen

As can be seen from Table 4, the detection specificity of the human immunodeficiency virus (HIV1+2) antibody detection kit of the reagent prepared by using the gp36-1 antigen is 100%, which indicates that the specificity of the gp36-1 antigen can meet the requirement of development of an immunodiagnostic reagent.

Example 5 thermostability assay for recombinant gp36 antigen

The gp36-1 antigen prepared in example 3 was heat treated at 37 deg.C for 7 days and 14 days, respectively, and the protein was not aggregated or degraded as detected by SDS-PAGE electrophoresis. The NC membrane was coated with the heat-treated antigen together with HIV-1 antigen (from Fipeng, Bio Inc.), and colloidal gold was labeled with HIV-1+2 antigen (from Fipeng, Bio Inc.) to prepare a human immunodeficiency virus (HIV1+2) antibody detection kit, which was used as a reference for a rapid reagent for detecting HIV antibody (see Table 5). The results show that the recombinant gp36 antigen of the invention has no significant differences in testing of the corporate reference samples after 7 and 14 days of heat treatment at 37 ℃ compared to freshly prepared antigen (see table 6).

TABLE 5 Enterprise reference Standard

TABLE 6 test results of heat-treated gp36-1 antigen test enterprise reference

Example 6 Freeze-thaw stability of recombinant gp36 antigen

The gp36-1 antigen prepared in example 3 was subjected to freeze-thawing 3 times and 5 times, respectively, and the protein was not aggregated or degraded as detected by SDS-PAGE electrophoresis. The antigen after freeze thawing and HIV-1 type antigen (from Fipeng Bio Inc.) are coated on an NC membrane at the same time, and the HIV-1+2 type antigen (from Fipeng Bio Inc.) is used for marking colloidal gold to prepare a human immunodeficiency virus (HIV1+2) antibody detection kit, so that the human immunodeficiency virus antibody rapid reagent enterprise reference product (shown in Table 5) is detected. The results show that the recombinant gp36 antigen of the invention has no significant difference in testing the corporate reference products after 3 and 5 times of freeze-thawing compared to freshly prepared antigen (see table 7).

TABLE 7 results of gp36 antigen testing enterprise reference after freeze-thawing

Example 7 sensitivity of recombinant gp36 antigen

NC membranes were coated with the gp36-1 antigen prepared in example 3 together with HIV-1 antigen (from Ficron, N.J.), colloidal gold was labeled with HIV-1+2 antigen (from Ficron, N.J.), and a human immunodeficiency virus (HIV1+2) antibody detection kit was prepared, and national references (see Table 8) as a rapid reagent for detecting human immunodeficiency virus antibodies were evaluated for sensitivity (see Table 9).

TABLE 8 national Standard of reference products

TABLE 9 national reference results for gp36-1 antigen detection

As can be seen from Table 9, the recombinant gp36-1 antigen of the present invention meets the requirements for national reference test of the rapid human immunodeficiency virus antibody reagents, indicating that the sensitivity of the antigen of the present invention can meet the requirements for development of immunodiagnostic reagents.

It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Sichuan Mike BioNew Material technology Co., Ltd

<120> HIV-2 recombinant antigen, preparation method and application thereof

<130> 2020

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 116

<212> PRT

<213> HIV-2

<400> 1

Gln Leu Leu Asp Val Val Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr

1 5 10 15

Val Trp Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys

20 25 30

Tyr Leu Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg

35 40 45

Gln Val Cys His Thr Thr Val Pro Trp Val Asn Asp Ser Leu Ala Pro

50 55 60

Asp Trp Asp Asn Met Thr Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr

65 70 75 80

Leu Glu Ala Asn Ile Ser Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln

85 90 95

Glu Lys Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Ile Phe

100 105 110

Gly Asn Trp Phe

115

<210> 2

<211> 131

<212> PRT

<213> HIV-2

<400> 2

Ala Gln Ser Arg Thr Leu Leu Ala Gly Ile Val Gln Gln Gln Gln Gln

1 5 10 15

Leu Leu Asp Val Val Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val

20 25 30

Trp Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr

35 40 45

Leu Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln

50 55 60

Val Cys His Thr Thr Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp

65 70 75 80

Trp Asp Asn Met Thr Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu

85 90 95

Glu Ala Asn Ile Ser Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu

100 105 110

Lys Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Ile Phe Gly

115 120 125

Asn Trp Phe

130

<210> 3

<211> 146

<212> PRT

<213> HIV-2

<400> 3

Ala Thr Ala Gly Ser Ala Met Gly Ala Ala Ser Leu Thr Val Ser Ala

1 5 10 15

Gln Ser Arg Thr Leu Leu Ala Gly Ile Val Gln Gln Gln Gln Gln Leu

20 25 30

Leu Asp Val Val Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val Trp

35 40 45

Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu

50 55 60

Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val

65 70 75 80

Cys His Thr Thr Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp

85 90 95

Asp Asn Met Thr Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu

100 105 110

Ala Asn Ile Ser Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys

115 120 125

Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Ile Phe Gly Asn

130 135 140

Trp Phe

145

<210> 4

<211> 160

<212> PRT

<213> HIV-2

<400> 4

Gly Phe Leu Ala Thr Ala Gly Ser Ala Met Gly Ala Ala Ser Leu Thr

1 5 10 15

Val Ser Ala Gln Ser Arg Thr Leu Leu Ala Gly Ile Val Gln Gln Gln

20 25 30

Gln Gln Leu Leu Asp Val Val Lys Arg Gln Gln Glu Leu Leu Arg Leu

35 40 45

Thr Val Trp Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu

50 55 60

Lys Tyr Leu Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe

65 70 75 80

Arg Gln Val Cys His Thr Thr Val Pro Trp Val Asn Asp Ser Leu Ala

85 90 95

Pro Asp Trp Asp Asn Met Thr Trp Gln Glu Trp Glu Lys Gln Val Arg

100 105 110

Tyr Leu Glu Ala Asn Ile Ser Lys Ser Leu Glu Gln Ala Gln Ile Gln

115 120 125

Gln Glu Lys Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Ile

130 135 140

Phe Gly Asn Trp Phe Asp Leu Thr Ser Trp Val Lys Tyr Ile Gln Tyr

145 150 155 160

<210> 5

<211> 348

<212> DNA

<213> Artificial Synthesis

<400> 5

cagctgctgg acgttgttaa acgtcagcag gaactgctgc gtctgaccgt ttggggtacc 60

aaaaacctgc aggctcgtgt taccgctatc gaaaaatacc tgcaggacca ggctcgtctg 120

aactcttggg gttgcgcttt ccgtcaggtt tgccacacca ccgttccgtg ggttaacgac 180

tctctggctc cggactggga caacatgacc tggcaggaat gggaaaaaca ggttcgttac 240

ctggaagcta acatctctaa atctctggaa caggctcaga tccagcagga aaaaaacatg 300

tacgaactgc agaaactgaa ctcttgggac atcttcggta actggttc 348

<210> 6

<211> 393

<212> DNA

<213> Artificial Synthesis

<400> 6

gctcagtctc gtaccctgct ggctggtatc gttcagcagc agcagcagct gctggacgtt 60

gttaaacgtc agcaggaact gctgcgtctg accgtttggg gtaccaaaaa cctgcaggct 120

cgtgttaccg ctatcgaaaa atacctgcag gaccaggctc gtctgaactc ttggggttgc 180

gctttccgtc aggtttgcca caccaccgtt ccgtgggtta acgactctct ggctccggac 240

tgggacaaca tgacctggca ggaatgggaa aaacaggttc gttacctgga agctaacatc 300

tctaaatctc tggaacaggc tcagatccag caggaaaaaa acatgtacga actgcagaaa 360

ctgaactctt gggacatctt cggtaactgg ttc 393

<210> 7

<211> 438

<212> DNA

<213> Artificial Synthesis

<400> 7

gctaccgctg gttctgctat gggtgctgct tctctgaccg tttctgctca gtctcgtacc 60

ctgctggctg gtatcgttca gcagcagcag cagctgctgg acgttgttaa acgtcagcag 120

gaactgctgc gtctgaccgt ttggggtacc aaaaacctgc aggctcgtgt taccgctatc 180

gaaaaatacc tgcaggacca ggctcgtctg aactcttggg gttgcgcttt ccgtcaggtt 240

tgccacacca ccgttccgtg ggttaacgac tctctggctc cggactggga caacatgacc 300

tggcaggaat gggaaaaaca ggttcgttac ctggaagcta acatctctaa atctctggaa 360

caggctcaga tccagcagga aaaaaacatg tacgaactgc agaaactgaa ctcttgggac 420

atcttcggta actggttc 438

<210> 8

<211> 480

<212> DNA

<213> Artificial Synthesis

<400> 8

ggtttcctgg ctaccgctgg ttctgctatg ggtgctgctt ctctgaccgt ttctgctcag 60

tctcgtaccc tgctggctgg tatcgttcag cagcagcagc agctgctgga cgttgttaaa 120

cgtcagcagg aactgctgcg tctgaccgtt tggggtacca aaaacctgca ggctcgtgtt 180

accgctatcg aaaaatacct gcaggaccag gctcgtctga actcttgggg ttgcgctttc 240

cgtcaggttt gccacaccac cgttccgtgg gttaacgact ctctggctcc ggactgggac 300

aacatgacct ggcaggaatg ggaaaaacag gttcgttacc tggaagctaa catctctaaa 360

tctctggaac aggctcagat ccagcaggaa aaaaacatgt acgaactgca gaaactgaac 420

tcttgggaca tcttcggtaa ctggttcgac ctgacctctt gggttaaata catccagtac 480

Claims

1. An HIV-2 recombinant antigen, the amino acid sequence of which is shown in SEQ ID NO. 1.

2. A polynucleotide, said polynucleotide:

a) encoding the recombinant antigen of claim 1;

b) as shown in SEQ ID NO. 5; or

c) Complementary to a) or b).

3. A polynucleotide construct comprising the polynucleotide of claim 2.

4. A host cell comprising the polynucleotide construct of claim 3 or the polynucleotide of claim 2.

5. A composition for detecting HIV comprising the recombinant antigen of claim 1.

6. The composition for detecting HIV of claim 5, wherein said recombinant antigen is additionally labeled with a detectable label or said recombinant antigen is coated on a solid support.

7. A kit comprising the recombinant antigen of claim 1.

8. The kit of claim 7, wherein,

1) the recombinant antigen is coated on a solid support;

2) the recombinant antigen is provided with a detectable label; or

3) The recombinant antigen is not coated on a solid support and is not detectably labeled.

9. Use of the recombinant antigen of claim 1 in the manufacture of a kit for the detection of HIV.

10. Use of a recombinant antigen according to claim 9 for the preparation of a kit for the detection of HIV, wherein said kit is used for the combined detection of HIV-1 and HIV-2.

11. A method of producing an HIV-2 recombinant antigen comprising:

1) introducing the polynucleotide of claim 2 into a host cell, or transferring the polynucleotide construct of claim 3 into a host cell;