WO2018119838A1 - Hiv重组抗原、表达基因、表达载体以及hiv检测试剂盒 - Google Patents

Hiv重组抗原、表达基因、表达载体以及hiv检测试剂盒 Download PDF

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WO2018119838A1
WO2018119838A1 PCT/CN2016/112933 CN2016112933W WO2018119838A1 WO 2018119838 A1 WO2018119838 A1 WO 2018119838A1 CN 2016112933 W CN2016112933 W CN 2016112933W WO 2018119838 A1 WO2018119838 A1 WO 2018119838A1
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hiv
fragment
monoclonal antibody
subtype
seq
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PCT/CN2016/112933
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French (fr)
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李瑞净
孟媛
于秀玲
王益琼
林育佳
孙康成
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菲鹏生物股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention relates to the field of biotechnology, in particular to an HIV recombinant antigen, an expression gene, an expression vector and an HIV detection kit.
  • HIV acquired immunodeficiency syndrome
  • methods for detecting AIDS at home and abroad include enzyme-linked immunosorbent assay, colloidal gold detection technology, immunoblot detection technology, nucleic acid detection technology and biochip detection technology.
  • Enzyme linked immunosorbent assay It is the most widely used technique in immunoassay. The basic method is to adsorb a known antigen on the surface of a solid phase carrier (polystyrene micro-reaction plate), wash the free component in the liquid phase by washing, add the sample, wash, add the enzyme-labeled antigen, and make the antigen-antibody The reaction is carried out on a solid surface for the determination of HIV-specific antibodies.
  • Colloidal-Gold Test is a new type of immunolabeling technology applied to antigen-antibody by using colloidal gold as a tracer marker. It can be used for fast screening, but has certain defects such as insufficient sensitivity.
  • Immunoblotting is a method for detecting a certain protein in a complex sample based on the specific binding of an antigen antibody. This method is a new immunological technique developed on the basis of gel electrophoresis and solid phase immunoassay. Due to Western blotting has the high specificity and sensitivity of SDS-PAGE for high resolution and solid phase immunoassays, and has now become a routine technique for protein analysis, but it is time consuming.
  • NAT Nucleic Acid Test
  • Bio-chip Test is a micro-biochemical analysis system that integrates discrete analysis processes in the life sciences field onto the surface of silicon chips or glass chips by microfilm technology based on the principle of specific interaction between molecules. In order to achieve accurate, rapid, and large information detection of cells, proteins, genes and other biological components, but expensive and not suitable for census.
  • ELISA has its own advantages and disadvantages. Because of its simple operation, good sensitivity and specificity, low cost and short detection time, ELISA has been widely used in medical structures such as blood stations, blood products factories and hospitals. It is the most widely used detection technology.
  • ELISA can be divided into indirect method, sandwich method, capture method, competition inhibition method and the like.
  • the development of the HIV enzyme-linked immunoassay reagent can be divided into four stages depending on the source of the antigen used in the reagent and the type of the detection reagent.
  • the first generation of reagents was introduced in 1985 using the principle of indirect methods, using lysates of HIV viruses cultured from T lymphocyte lines, which often contain contamination of host cell components, causing false positives.
  • the most common component of the contaminating component is the protein fragment derived from the major histocompatibility region of the virus-spreading lymphocytes. Therefore, individuals exposed to exogenous HLA due to exposure to embryonic leukocytes during pregnancy or due to blood transfusion have anti-HLA antibodies. Sometimes there are false positives. In order to reduce non-specificity, it is usually necessary to reduce the concentration of the coated antigen. In addition, the presence of the heteroprotein competes with the antigen for the coating site, and thus the sensitivity of the first-generation reagent is also limited.
  • the second generation of reagents in order to improve sensitivity and specificity, began to use genetic engineering recombination and/or synthetic peptides as antigens, and the use of recombinant antigens and synthetic peptides significantly increased the proportion of immunodominant regions in the coated antigens through genetic recombination.
  • the method is generally easy to produce a large amount of homologous antigen in a medium, and the expression product is a single target gene rather than a mixture of diversity antigens, thereby improving the sensitivity of the reagent, Moreover, the purity of the antigen produced under standardized conditions is higher, and the specificity and repeatability of the reagent are correspondingly increased.
  • the second-generation reagents still use the indirect method, which first coats the polystyrene plate with the antigen, adds the serum to be tested, and finally adds the enzyme-labeled anti-human IgG antibody, so that only the sample can be detected.
  • the IgG antibody in the medium limits the detection of the reagent for the HIV infection window.
  • the third-generation reagent uses synthetic peptide or recombinant HIV antigen, and uses the double antigen sandwich principle to detect HIV antibody, that is, the antigen is coated with polystyrene plate, and then the serum to be tested and the enzyme-labeled antigen are added, so that all types can be detected simultaneously.
  • the antibody increases the sensitivity of the reagent while the specificity is also improved.
  • the third-generation reagent can detect IgM antibodies, it can serve to shorten the window period. It has been reported that the third-generation HIV antibody detection reagents have an average window detection time of 5 days earlier than the second generation products.
  • the fourth generation reagent is a combined detection reagent for HIV-1 P24 antigen and HIV antibody.
  • the reagents are coated with the HIV antigen and the anti-p24 antibody simultaneously on the polystyrene plate, then the biotin-labeled P24 antibody and the serum to be tested are added, and finally the enzyme-labeled antigen and streptavidin are added, thereby Simultaneous detection of antibodies and antigens in serum.
  • This reagent can detect early seroconversion and further improve the safety of blood transfusion.
  • the window period for the detection of HIV enzymes in the detection community has been: first generation 6 weeks, second generation 5 weeks, third generation 3 weeks, fourth generation 2 weeks, correspondingly reduce the residual risk of blood transfusion
  • the fourth-generation reagents for large-scale blood donor screening can improve the safety of blood transfusion and have obvious detection advantages.
  • the third-generation double-antigen sandwich reagent is the most commonly used in clinical and blood screening, but with its continuous maturity and improvement, the fourth-generation reagent will gradually be used as a mainstream reagent for blood screening.
  • HIV testing reagents currently only target HIV-1 subtype antibodies and HIV-2 subtype antibodies, and do not target HIV-O subtype antibodies. Although HIV-O subtypes are rarely distributed in China, with the increasing mobility of domestic and foreign personnel, China's HIV-O subtype infection has a tendency to strengthen; on the other hand, domestic HIV testing reagents will also be exported to HIV-O. Sub-type infections are more serious in countries and regions, so the risk of missed detection of HIV-O subtypes by existing reagents is increasing.
  • An HIV recombinant antigen comprising a HIV-1 subtype segment, an HIV-2 subtype segment, and an HIV-O subtype segment;
  • the HIV-O subtype fragment is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 3.
  • An expression gene comprising a HIV-1 fragment, an HIV-2 fragment, and an HIV-O fragment
  • the sequence of the HIV-O fragment is shown in SEQ ID No. 3.
  • An expression vector comprising the above expressed gene.
  • An HIV detection kit comprising the above-mentioned HIV recombinant antigen, a marker-labeled HIV recombinant antigen, a first anti-P24 monoclonal antibody, a second anti-P24 monoclonal antibody, and a third anti-P24 monoclonal antibody.
  • This HIV recombinant antigen contains HIV-O fragments and can be used to detect HIV-O subtypes.
  • HIV detection kits including such HIV recombinant antigens exhibit higher specificity and sensitivity in detecting HIV-O subtypes.
  • Example 1 is a SDS-PAGE electrophoresis pattern of the coated antigen and the labeled antigen prepared in Example 1;
  • Example 2 is a SDS-PAGE electrophoresis pattern of three anti-P24 monoclonal antibodies purified from ascites of mice prepared from three strains of P24 hybridoma cells in Example 2.
  • An HIV recombinant antigen of one embodiment comprises a HIV-1 subtype segment, an HIV-2 subtype segment, and an HIV-O subtype segment, which are ligated in sequence.
  • the HIV-1 subtype is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 1, or the HIV-1 subtype is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 4. .
  • the HIV-2 subtype fragment is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 2.
  • the HIV-O subtype fragment is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 3.
  • Expression genes for expression of the above HIV recombinant antigen include HIV-1 fragments, HIV-2 fragments and HIV-O fragments which are ligated in sequence.
  • the sequence of the HIV-1 fragment is shown in SEQ ID No. 1, or the sequence of the HIV-1 fragment is shown in SEQ ID No. 4.
  • the sequence of the HIV-2 fragment is shown in SEQ ID No. 2.
  • the sequence of the HIV-O fragment is shown in SEQ ID No. 3.
  • the nucleotide sequence shown by SEQ ID No. 1 is referred to as H I
  • the nucleotide sequence represented by SEQ ID No. 2 is designated as H II
  • the nucleotide sequence represented by SEQ ID No. 3 is recorded.
  • the nucleotide sequence shown by SEQ ID No. 4 is referred to as H I A.
  • the above gene fragments are genetically engineered techniques and analyzed by a large number of molecular biology analysis software to screen out the dominant epitope gene segments.
  • H I is a dominant epitope gene segment of the gp41 protein of the HIV-1 subtype.
  • H II is a dominant epitope gene segment of the gp36 protein of the HIV-2 subtype.
  • O is a dominant epitope gene segment of the gp41 protein of the HIV-O subtype.
  • H I A is a fragment obtained by point mutation of H I , and the mutation site is T67A and D121E of the gp41 protein of the HIV-1 subtype.
  • Expression vectors comprising the above expressed genes are also disclosed.
  • the expression vector comprising the above expressed gene may be pET-24a (+) or PGEX4T-1.
  • the expression vector for expressing the gene is pET-24a(+)
  • the above-mentioned HIV is expressed.
  • the recombinant antigen has a His-tag at the end of the HIV-O subtype that is remote from the HIV-1 type.
  • the expression vector for expressing the gene is PGEX4T-1
  • the above-mentioned HIV recombinant antigen expressed in the HIV-1 type segment has GST at the end away from the HIV-O subtype segment.
  • An expression vector comprising the above expressed gene (exemplified by H I AH II-O) can be obtained by designing a primer to amplify a target fragment according to the sequence of the above expressed gene, and the upstream primer of the H I A fragment carries a BamHI site, downstream.
  • the primers carry the EcoRI restriction site
  • the upstream primer of the H II fragment carries the EcoRI site
  • the downstream primer carries the SalI restriction site
  • the upstream primer of the O fragment carries the SalI site
  • the downstream primer carries the XhoI restriction site.
  • the PCR fragment was digested with the corresponding restriction endonuclease, and the H I A fragment was ligated into the expression vector pET-24a(+) digested with BamHI and EcoRI to obtain the recombinant plasmid pET-24a(+)- H I A, then pET-24a(+)-H I A was digested with EcoRI and SalI, and the H II fragment was ligated, and then pET-24a(+)-H I AH II was digested with SalI and XhoI. The fragment was ligated into E.
  • coli expression strain BL21(DE3)pLysS to obtain an expression vector pET-24a(+)-H I AH II-O including the above expressed gene.
  • the recombinant plasmid pET-24a(+)-H I AH II-O was digested with BamHI and XhoI, and the fragment H I AH II-O was excised, ligated into the vector PGEX4T-1, and transformed into E. coli expression strain.
  • BL21(DE3)pLysS an expression vector PGEX4T-1-H I AH II-O including the above expressed gene was obtained.
  • the expression vector pET-24a(+)-H I A-H II-O including the above-described expression gene and the expression vector PGEX4T-1-H I A-H II-O including the above-described expression gene can be used for the expression of the above-described HIV recombinant antigen.
  • HIV recombinant antigen can be applied to the field of HIV detection, and the following is a brief introduction to the application of the HIV detection kit.
  • the invention discloses an HIV detection kit according to an embodiment, comprising the above HIV recombinant antigen, the labeled HIV recombinant antigen labeled with the marker, the first anti-P24 monoclonal antibody, the second anti-P24 monoclonal antibody and the third anti-P24 monoclonal antibody.
  • the HIV detection kit is a fourth-generation HIV detection reagent, and the recombinant antigen expressed by the above pET-24a(+)-H I AH II-O is used as a coating antigen, and PGEX4T-1-H I AH II-O is expressed.
  • the recombinant antigen acts as a labeled antigen.
  • the label can be HRP.
  • the first anti-P24 monoclonal antibody, the second anti-P24 monoclonal antibody are coated antibodies, and the third anti-P24 monoclonal antibody is a labeled antibody.
  • the first anti-P24 monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line P24-2F4.
  • the hybridoma cell line P24-2F4 was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No: C2016211, classification: Hybridoma cell line P24-2F4.
  • the second anti-P24 monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line P24-5C2.
  • the hybridoma cell line P24-5C2 was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No: C2016212, classification: Hybridoma cell line P24-5C2.
  • the third anti-P24 monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line P24-3B9.
  • the hybridoma cell line P24-3B9 was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No: C2016213, classification: Hybridoma cell line P24-3B9.
  • the sequence is SEQ ID No. .1 (designated H I), SEQ ID No. 2 (designated H II), SEQ ID No. 3 (designated O), designed primer amplification target fragment, and H I fragment for specific kit specificity Point mutations, the mutation sites were HIV-1 gp41T67A and D121E, and the mutated sequence was named H I A (SEQ ID No. 4).
  • the upstream primer of the H I A fragment carries a BamHI site, a downstream primer With the EcoRI restriction site, the upstream primer of the H II fragment carries the EcoRI site, the downstream primer carries the SalI restriction site, the upstream primer of the O fragment carries the SalI site, and the downstream primer carries the XhoI restriction site.
  • PCR The fragment was digested with the corresponding restriction endonuclease, and the H I A fragment was ligated into the expression vector pET-24a(+) digested with BamHI and EcoRI to obtain the recombinant plasmid pET-24a(+)-H.
  • the recombinant plasmid pET-24a(+)-H I AH II-O was digested with BamHI and XhoI, and the fragment H I AH II-O was excised, ligated into the vector PGEX4T-1, and transformed into E. coli expression strain.
  • BL21(DE3)pLysS an expression vector PGEX4T-1-H I AH II-O including the above expressed gene was obtained.
  • BL21(DE3)pLysS strain containing expression vector pET-24a(+)-H I AH II-O is used with 100 ⁇ g/mL kanamycin sulfate (Shanghai Shenggong Bioengineering Technology Service Co., Ltd., hereinafter referred to as the labor, article number A500636) 500 mL of LB medium was shake-cultured at 37 ° C to an OD600 of about 1.0, and induced with IPTG (Biotech, Cat. No. A100487) at a final concentration of 0.25 mM for 6 hours at 28 °C. The cells were collected by centrifugation at 7000 rpm for 3 minutes at 4 ° C.
  • the cells of each liter of bacteria were resuspended in 20 mL of lysis buffer (50 mM Tirs-HCl, pH 8.0, 500 mM NaCl), sonicated, centrifuged at 12000 g for 4 minutes at 4 ° C, and collected.
  • the NI column was cleaned (Buffer A: 50 mM Tirs-HCl, 500 mM NaCl pH 8.0; Buffer B: 50 mM Tirs-HCl, 500 mM NaCl, 200 mM imidazole, pH 8.0).
  • the target protein which is designated as H I AH II-O-His-tag, is used as a coating antigen, as shown in Figure 1, lane 2, and stored at -20 °C for later use.
  • IPTG Biotech, Cat. No. A100487 with a final concentration of 0.5 mM was induced for 4 hours at 37 °C.
  • the cells were collected by centrifugation at 7000 rpm for 3 minutes at 4 ° C, and 20 mL of cells per liter of the bacterial solution was used.
  • the suspension buffer (50 mM Tirs-HCl, 500 mM NaCl, pH 8.0) was resuspended, sonicated, centrifuged at 12000 g for 20 minutes at 4 ° C, and the supernatant was collected through a GST column (Buffer A: 20 mM PB + 150 mM NaCl, Ph 7.4; Buffer B: 25 mM Tirs-HCl + 10 m MGSH, pH 8.0).
  • the target protein which is designated as GST-H I AH II-O, is used as a labeled antigen, as shown in Figure 1, lane 3, and stored at -20 °C for later use.
  • the P24 recombinant antigen (P24-Ag, Philippine Biotech Co., Ltd.) was diluted to 1.0 mg/mL, mixed with Freund's complete adjuvant (Sigma-Aldrich, product number: F5881) in an equal volume, and fully emulsified to obtain an oily emulsion. .
  • the emulsion was subcutaneously administered to BALB/c mice at a dose of 0.2 mL (Guangdong Medical Laboratory Animal Center: No. 119, Huangqiyang Road, Nanhai, Foshan City, Guangdongzhou, 6-week-old female, 5) back sites.
  • the abdominal cavity was boosted, that is, the same amount of antigen was mixed with the Freund's incomplete adjuvant (Sigma-Aldrich, F5506) in the same volume, and the immunization was boosted to four needles.
  • the tail blood was collected and the serum was separated for indirect ELISA.
  • the titer is determined by the method, and the titer is higher than 1:10000 for fusion.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • the BALB/c mice were sacrificed by neck-stretching, 75% alcohol was immersed in the whole body, and the abdominal skin was cut with scissors under aseptic operation.
  • the peritoneum was exposed, and 5 mL of RPMI 1640 basic culture solution was injected into the abdominal cavity with a syringe. Rinse repeatedly, recover the rinse solution, centrifuge at 1000 rpm for 5 minutes, leave a precipitate, resuspend with RPMI 1640 screening medium (in RPMI 1640 complete medium containing HAT), adjust the cell concentration to 1 ⁇ 10 5 /mL, and add 96 wells. Plates, 150 ⁇ L/well, 37 ° C, 5% CO 2 were incubated overnight.
  • mice Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a dish, rinsed once with RPMI 1640 base medium, and placed in a nylon beaker on a small beaker to be filtered to prepare a cell suspension. After centrifugation, the supernatant was discarded, and the RPMI 1640 base medium was resuspended, and this was repeated three times and counted.
  • Mouse myeloma cells Sp2/0 (preserved by Fei Peng Biotechnology Co., Ltd.) were screened by 8-azaguanine and cultured to logarithmic growth phase. Two large bottles were prepared to make cell suspension, centrifuged, and the supernatant was discarded. RPMI1640 was used. The base medium was resuspended and repeated three times, counting.
  • the myeloma cells and the immune spleen cells were mixed at a ratio of 1:10, and washed once with a RPMI 1640 base culture solution in a 50 mL plastic centrifuge tube, and centrifuged at 1200 rpm for 8 minutes. The supernatant was discarded, the cells were mixed, 1 mL of 50% PEG1500 fusion was slowly added, and after 1 minute of fusion, 15 mL of RPMI1640 basal medium was added to terminate the cell fusion. Centrifuge for 5 minutes at 1000 rpm.
  • the supernatant was discarded, and gently incubated with 50 mL of RPMI 1640 screening medium, aliquoted into 10 96-well plates plated with feeder cells, 50 ⁇ L/well, and cultured at 37 ° C, 5% CO 2 . The culture was continued until the sixth day, and the HT medium (HTMI-containing RPMI1640 complete medium) was changed twice.
  • the P24-Ag protein was diluted with 0.06 M pH 9.6 carbonate buffer solution to a final concentration of 2 ⁇ g/mL. 0.1 mL per well was added to a 96-well polystyrene plate and incubated at 37 ° C for 2 hours or 4 ° C overnight. The next day, it was blocked with 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder at 0.15 mL/well for 2 hours at 37 ° C for detection.
  • hybridoma cell line P24-2F4 Three hybridoma cell deposits were designated as hybridoma cell line P24-5C2 and hybridoma cell line P24-3B9.
  • hybridoma cell line P24-2F4 was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No: C2016211, classification: Hybridoma cell line P24-2F4.
  • hybridoma cell line P24-5C2 was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No: C2016212, classification: Hybridoma cell line P24-5C2.
  • hybridoma cell line P24-3B9 was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No: C2016213, classification: Hybridoma cell line P24-3B9.
  • the monoclonal antibody secreted by the hybridoma cell line P24-2F4 was recorded as the first anti-P24 monoclonal antibody
  • the monoclonal antibody secreted by the hybridoma cell line P24-5C2 was recorded as the second anti-P24 monoclonal antibody
  • the hybridoma cell line P24- The monoclonal antibody secreted by 3B9 was recorded as the third anti-P24 monoclonal antibody.
  • mice (Guangdong Medical Laboratory Animal Center: No. 119, Huangqiyang Road, Nanhai, Foshan City, Guangdong province, 6-week-old female), each mouse was intraperitoneally injected with 0.5 mL of phytane; 10 days later 1 ⁇ 10 6 P24-2F4 hybridoma cells, 1 ⁇ 10 6 P24-5C2 hybridoma cells, and 1 ⁇ 10 6 P24-3B9 hybridoma cells were intraperitoneally injected. After inoculation of cells for 7 to 10 days, ascites can be produced, and the animal's health and ascites signs should be closely observed.
  • mice As much as possible of ascites, while the mice are killed before death, the mice are sacrificed and the ascites is inhaled into the test tube with a dropper.
  • the mice can obtain 5-10 mL of ascites.
  • Ascites was collected, centrifuged, and diluted with 3 volumes of PBS and filtered through a filter paper. The resulting filtrate was applied to a protein G affinity chromatography column (GE) equilibrated with PBS at a flow rate of 1 mL/min. The material not adsorbed by protein G was then washed with PBS at a flow rate of 1 mL/min until the adsorption value at OD280 nm reached the baseline.
  • GE protein G affinity chromatography column
  • the antibody was eluted and recovered with a 0.1 M glycine eluate (pH 2.5). The recovered antibody was immediately neutralized with 1.5 M Tris (pH 8.8), and the SDS-PAGE gel was run, as shown in Fig. 2, wherein lane 1 was a marker, and lanes 2 to 4 were purified anti-P24 monoclonal antibodies. As can be seen from Figure 2, the purity of the three anti-P24 monoclonal antibodies was above 95%.
  • the purified monoclonal antibody to be identified was diluted with 0.06 M pH 9.6 carbonate buffer solution to a final concentration of 1 ⁇ g/mL. 0.1 mL per well was added to a 96-well polystyrene plate and incubated at 37 ° C for 2 hours or 4 ° C overnight. The next day, 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder, 0.15 mL/well, blocked at 37 ° C for 2 hours, and 2000-fold diluted horseradish peroxidase-labeled P24 epitope was added to identify the antigen.
  • the NaIO 4 oxidation method was employed. Weigh 8mg horseradish peroxidase (HRP, British Biozyme laboratories company, article number: HRP4) dissolved in 0.4mL ultrapure water, and slowly add 0.4mL ultrapure water freshly prepared 20mg/mL NaIO 4 solution, room temperature After gently stirring for 40 minutes under light, a solution of 48 ⁇ L of ethylene glycol (8 ⁇ L of ethylene glycol dissolved in 40 ⁇ L of distilled water) was added, and the mixture was stirred at room temperature for 40 minutes in the dark.
  • HRP4 horseradish peroxidase
  • the third anti-P24 monoclonal antibody obtained in Example 2 was diluted to 1 mg/mL, and dialyzed against 20 mM PB pH 7.4 for 24 h; biotin was dissolved in H 2 O to prepare 10 mM/L; The cells were added to a dialysis bag and dialyzed against PB pH 7.4 dialysate for 12 hours.
  • the biotin-labeled third anti-P24 monoclonal antibody was added to a final concentration of 15% NBS and 50% glycerol for storage.
  • the fourth generation ELISA detection method of the present invention is established.
  • Coating The coated antigen H I AH II-O-Histag, the first anti-P24 monoclonal antibody and the second anti-P24 monoclonal antibody obtained in Example 1 were mass ratio 1:10:10 (0.2 ⁇ g: 2 ⁇ g: 2 ⁇ g) was added to 50 mM CB of pH 9.6, mixed for coating, 100 ⁇ L per well, and coated at 4 ° C overnight (about 22 h);
  • Blocking Take the coated plate from 4 °C, equilibrate to room temperature, wash the plate twice; add 120 ⁇ L blocking solution to each well, block overnight at 4 °C (about 20 h) or block at 37 °C for 2 h; stand-by;
  • Biotin-labeled third anti-P24 monoclonal antibody was added to the biotin dilution at a volume ratio of 1:1000 and mixed. First, 25 ⁇ L of P24 biotin labeling reagent was added to each well, and then 75 ⁇ L of the sample to be tested, negative and positive control were added to the corresponding wells, and incubated at 37 ° C for 60 min.
  • Washing discard the liquid in the well plate, wash the plate with the washing solution 5 times, soak for 30 seconds each time;
  • Washing discard the liquid in the well plate, wash the plate with the washing solution 5 times, soak for 30 seconds each time;
  • Termination add 50 ⁇ L of stop solution to each well and mix gently by shaking;
  • Reading value The OD value was read at 450 nm and 630 nm on a microplate reader.
  • Example 3 It can be seen from Table 1 and Table 2 that the HIV detection kit obtained in Example 3 has certain advantages in sensitivity and specificity with similar products at home and abroad.
  • HIV-O specimens purchased from the National Institute for Biological Standards and Control (NIBSC) were tested and the results (OD) Values are shown in Table 3 below.
  • HIV-O specimen II 1.021 0.988 0.175 HIV-O specimen III 0.665 0.142 0.115
  • the HIV detection kit obtained in Example 3 has a slight advantage over the existing well-known foreign companies A, and has a significant advantage over the domestic company B.
  • the HIV detection kit obtained in Example 3 has higher sensitivity than the existing products in the detection of the P24, and may prevent missed detection.

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Abstract

一种能够用于检测HIV-O亚型的HIV重组抗原，表达该HIV重组抗原的表达基因和表达载体，以及含有该HIV重组抗原的HIV检测试剂盒。HIV重组抗原，包括依次连接的HIV-1型段、HIV-2型段和HIV-O亚型段；所述HIV-O亚型段为由SEQ ID No.3所示的核苷酸序列编码得到的多肽。

Description

HIV重组抗原、表达基因、表达载体以及HIV检测试剂盒 技术领域：
本发明涉及生物技术领域，特别是涉及一种HIV重组抗原、表达基因、表达载体以及HIV检测试剂盒。
背景技术：
自1982年命名获得性免疫缺陷病综合症(AIDS)以来，HIV(Human immunodeficiency virus)的感染在全球广泛流行，严重威胁着人们的生命健康，因此，必须对HIV感染进行监控。有鉴于此，研发简便、准确、快速的检测产品，及早检测艾滋病病毒感染，杜绝艾滋病的传播成为预防控制艾滋病的首要手段。
目前国内外用于检测艾滋病的方法有酶联免疫吸附实验、胶体金检测技术、免疫印迹检测技术、核酸检测技术和生物芯片检测技术。
酶联免疫吸附实验(enzyme linked immunosorbent assay，ELISA)：是免疫测定技术中应用最广的技术。其基本方法是将已知的抗原吸附在固相载体(聚苯乙烯微量反应板)表面，用洗涤法将液相中的游离成分洗除，加入标本，洗涤，加入酶标记抗原，使抗原抗体反应在固相表面进行，用于测定HIV特异抗体。
胶体金检测技术(Colloidal-Gold Test)是以胶体金作为示踪标志物应用于抗原抗体的一种新型的免疫标记技术，能够用于快筛查，但有一定的缺陷如灵敏度不够等等。
免疫印迹检测技术(Immunoblotting)又称蛋白质印迹(Western blotting)，是根据抗原抗体的特异性结合检测复杂样品中的某种蛋白的方法。该方法是在凝胶电泳和固相免疫测定技术基础上发展起来的一种新的免疫技术。由于 免疫印迹具有SDS-PAGE的高分辨力和固相免疫测定的高特异性和敏感性，现已成为蛋白分析的一种常规技术，但比较耗时。
核酸检测技术(Nucleic Acid Test，NAT)是利用分子生物学的理论和技术，它的目标分子是DNA或RNA，通过直接探查病毒核酸的存在与否，从而对人体状态与疾病做出诊断的方法，但其对标本及核酸扩增酶的灵敏度及特异性要求较高，目前国内外还没有大量普及。
生物芯片检测技术(Bio-chip Test)是通过缩微技术，根据分子间特异性地相互作用的原理，将生命科学领域中不连续的分析过程集成于硅芯片或玻璃芯片表面的微型生物化学分析系统，以实现对细胞、蛋白质、基因及其它生物组分的准确、快速、大信息量的检测，但价格昂贵不适合普查。
每种检测技术各有优缺，ELISA检测因为其操作简单、灵敏度和特异性好、成本低廉、检测时间短等特点，已经被各大血站、血液制品厂、医院等医疗结构所普遍采用，是应用最广的一项检测技术。
按照检测原理，ELISA可以分为间接法、夹心法、捕获法、竞争抑制法等。根据试剂所用抗原的来源以及检测试剂的类型，可将HIV酶联免疫检测试剂的发展分为4个阶段。
第一代试剂问世于1985年，采用间接法原理，使用来自T淋巴细胞系中培养的HIV病毒的裂解产物，这类抗原通常会同时含有宿主细胞成分的污染，从而造成假阳性。其污染成分最常见的是病毒寄生的淋巴细胞的主要组织相容性区域来源的蛋白质片段，因此，因在怀孕期间暴露于胚胎白细胞或由于输血而与外源HLA接触的个体由于具有抗HLA抗体而有时会出现假阳性。为减少非特异性，通常不得不降低包被的抗原浓度，另外，杂蛋白的存在会与抗原竞争包被位点，因此第一代试剂的灵敏度也受到了一定的限制。
第二代试剂为提高灵敏度和特异度，开始使用基因工程重组和(或)人工合成肽作为抗原，重组抗原和合成肽的使用使包被抗原中免疫优势区的比例明显增强，通过基因重组的方法，通常易于在培养基中生产大量同源性抗原，表达产物为单一目的基因而非多样性抗原混合，从而提高了试剂的灵敏度， 而且在标准化条件下生产的抗原纯度更高，使试剂的特异性和重复性均相应提高。但和第一代试剂一样，第二代试剂仍然使用间接法原理，即先以抗原包被聚苯乙烯板，再加入待检血清，最后加入酶标记的抗人IgG抗体，因而只能检测样品中的IgG抗体，限制了试剂对HIV感染窗口期的检测。
第三代试剂使用合成多肽或重组HIV抗原，应用双抗原夹心法原理检测HIV抗体，即先以抗原包被聚苯乙烯板，再加入待检血清、酶标记的抗原，从而可同时检测所有类型抗体，提高了试剂的敏感性，同时特异性也得到了提高。另外，由于第三代试剂能检出IgM抗体，因此可以起到缩短窗口期的作用。有报道称，与第二代产品相比，第三代HIV抗体检测试剂检出窗口期的时间平均可提前5天。
第四代试剂为HIV-1P24抗原和HIV抗体联合检测试剂。为进一步缩短窗口期，此类试剂将HIV抗原和抗p24抗体同时包被聚苯乙烯板，再加入生物素标记P24抗体及待检血清，最后加入酶标记的抗原和链霉亲和素，从而能同时检测出血清中的抗体和抗原。这种试剂能够检测早期的血清阳转，进一步提高输血的安全性。检测界对HIV各代酶联检测试剂的窗口期一直是：第一代6周，第二代5周，第三代3周，第四代2周，相应地使输血的残余危险度进一步降低，第四代试剂用于大规模献血员筛查可以提高输血安全性，具有明显的检测优势。目前在临床和血液筛查中使用最为普遍的是第三代双抗原夹心法试剂，但随着自身的不断成熟和完善，第四代试剂将逐步作为主流试剂用于血液筛查。
国内艾滋病检测试剂目前基本上只针对HIV-1亚型抗体和HIV-2亚型抗体，并没有针对HIV-O亚型抗体。虽然HIV-O亚型在中国分布极少，但是随着国内外人员流动的不断加强，中国HIV-O亚型感染有加强的趋势；另一方面，国内艾滋病检测试剂也会出口到HIV-O亚型感染较为严重的国家和地区，所以现有试剂对HIV-O亚型的漏检风险越来越大。
发明内容：
基于此，有必要提供一种能够用于检测HIV-O亚型的HIV重组抗原。
此外还有必要提供用于表达上述HIV重组抗原的表达基因和表达载体，以及含有上述HIV重组抗原的HIV检测试剂盒。
一种HIV重组抗原，包括依次连接的HIV-1亚型段、HIV-2亚型段和HIV-O亚型段；
所述HIV-O亚型段为由SEQ ID No.3所示的核苷酸序列编码得到的多肽。
一种表达基因，包括依次连接的HIV-1片段、HIV-2片段和HIV-O片段；
所述HIV-O片段的序列如SEQ ID No.3所示。
一种表达载体，包括上述的表达基因。
一种HIV检测试剂盒，其特征在于，包括上述的HIV重组抗原、标记物标记的所述HIV重组抗原、第一抗P24单克隆抗体、第二抗P24单克隆抗体和第三抗P24单克隆抗体。
这种HIV重组抗原中含有HIV-O片段，从而可以用于检测HIV-O亚型。在具体的试验中，包括这种HIV重组抗原的HIV检测试剂盒在检测HIV-O亚型时表现出较高的特异性和灵敏性。
附图说明
图1为实施例1制得的包被抗原以及标记抗原的SDS-PAGE电泳图；
图2为实施例2中由三株P24杂交瘤细胞制备的小鼠腹水纯化得到的三株抗P24单克隆抗体的SDS-PAGE电泳图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似 改进，因此本发明不受下面公开的具体实施的限制。
一实施方式的HIV重组抗原，包括依次连接的HIV-1亚型段、HIV-2亚型段和HIV-O亚型段。
HIV-1亚型段为由SEQ ID No.1所示的核苷酸序列编码得到的多肽，或者HIV-1亚型段为由SEQ ID No.4所示的核苷酸序列编码得到的多肽。
HIV-2亚型段为由SEQ ID No.2所示的核苷酸序列编码得到的多肽。
HIV-O亚型段为由SEQ ID No.3所示的核苷酸序列编码得到的多肽。
还公开了用于表达上述HIV重组抗原的表达基因。
用于表达上述HIV重组抗原的表达基因包括依次连接的HIV-1片段、HIV-2片段和HIV-O片段。
HIV-1片段的序列如SEQ ID No.1所示，或者HIV-1片段的序列如SEQ ID No.4所示。
HIV-2片段的序列如SEQ ID No.2所示。
HIV-O片段的序列如SEQ ID No.3所示。
由SEQ ID No.1所示的核苷酸序列记为H I，由SEQ ID No.2所示的核苷酸序列记为H II，由SEQ ID No.3所示的核苷酸序列记为O，由SEQ ID No.4所示的核苷酸序列记为H I A。
上述基因片段为采用基因工程技术手段，通过大量分子生物学分析软件分析筛选出来的优势表位基因区段。
H I为HIV-1亚型的gp41蛋白的优势表位基因区段。
H II为HIV-2亚型的gp36蛋白的优势表位基因区段。
O为HIV-O亚型的gp41蛋白的优势表位基因区段。
H I A为H I进行点突变得到的片段，突变位点为HIV-1亚型的gp41蛋白的T67A和D121E。
还公开了包括上述表达基因的表达载体。
包括上述表达基因的表达载体可以为pET-24a(+)或PGEX4T-1。
具体的，上述表达基因的表达载体为pET-24a(+)时，表达得到的上述HIV 重组抗原在HIV-O亚型段远离HIV-1型段的一端有His-tag。
具体的，上述表达基因的表达载体为PGEX4T-1时，表达得到的上述HIV重组抗原在HIV-1型段远离HIV-O亚型段的一端有GST。
包括上述表达基因(以H I A-H II-O为例)的表达载体可以通过如下操作得到：按照上述表达基因的序列设计引物扩增目的片段，H I A片段上游引物带有BamHI位点，下游引物带有EcoRI酶切位点，H II片段上游引物带有EcoRI位点，下游引物带有SalI酶切位点，O片段上游引物带有SalI位点，下游引物带有XhoI酶切位点，PCR的片段经回收用相应的限制性内切酶酶切，H I A片段连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(+)中，得到重组质粒pET-24a(+)-H I A，之后用EcoRI及SalI酶切pET-24a(+)-H I A，将H II片段连入，之后用SalI及XhoI酶切pET-24a(+)-H I A-H II，将O片段连入，转入大肠杆菌表达菌株BL21(DE3)pLysS，得到包括上述表达基因的表达载体pET-24a(+)-H I A-H II-O。经测序正确后用BamHI及XhoI酶切重组质粒pET-24a(+)-H I A-H II-O，切下片段H I A-H II-O，连入载体PGEX4T-1中，转入大肠杆菌表达菌株BL21(DE3)pLysS，得到包括上述表达基因的表达载体PGEX4T-1-H I A-H II-O。
包括上述表达基因的表达载体pET-24a(+)-H I A-H II-O以及包括上述表达基因的表达载体PGEX4T-1-H I A-H II-O均可以用于上述HIV重组抗原的表达。
上述HIV重组抗原可以应用于HIV检测领域，下面以应用于HIV检测试剂盒为例进行简单介绍。
本发明公开了一实施方式的HIV检测试剂盒，包括上述HIV重组抗原、标记物标记的上述HIV重组抗原、第一抗P24单克隆抗体、第二抗P24单克隆抗体和第三抗P24单克隆抗体。
这种HIV检测试剂盒为第四代HIV检测试剂，上述pET-24a(+)-H I A-H II-O表达出来的重组抗原作为包被抗原，PGEX4T-1-H I A-H II-O表达出来的重组抗原作为标记抗原。
标记物可以为HRP。
第一抗P24单克隆抗体、第二抗P24单克隆抗体为包被抗体，第三抗P24单克隆抗体为标记抗体。
具体的，第一抗P24单克隆抗体为杂交瘤细胞株P24-2F4分泌的单克隆抗体。杂交瘤细胞株P24-2F4于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016211，分类命名：杂交瘤细胞株P24-2F4。
具体的，第二抗P24单克隆抗体为杂交瘤细胞株P24-5C2分泌的单克隆抗体。杂交瘤细胞株P24-5C2于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016212，分类命名：杂交瘤细胞株P24-5C2。
具体的，第三抗P24单克隆抗体为杂交瘤细胞株P24-3B9分泌的单克隆抗体。杂交瘤细胞株P24-3B9于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016213，分类命名：杂交瘤细胞株P24-3B9。
以下为具体实施例。
实施例中采用药物和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，通常按照常规条件，例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。
实施例1、包被抗原及标记抗原的制备
采用基因工程技术手段，通过大量的分子生物学分析软件分析筛选出HIV-1的gp41蛋白，O亚型的gp41蛋白，HIV-2的gp36蛋白的优势表位基因区段，序列为SEQ ID No.1(命名为H I)，SEQ ID No.2(命名为H II)，SEQ ID No.3(命名为O)，设计引物扩增目的片段，为提高试剂盒特异性对H I片段进行点突变，突变位点为HIV-1gp41T67A及D121E，突变后的序列命名为H I A(SEQ ID No.4)。H I A片段上游引物带有BamHI位点，下游引物 带有EcoRI酶切位点，H II片段上游引物带有EcoRI位点，下游引物带有SalI酶切位点，O片段上游引物带有SalI位点，下游引物带有XhoI酶切位点，PCR的片段经回收用相应的限制性内切酶酶切，H I A片段连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(+)中，得到重组质粒pET-24a(+)-H I A，之后用EcoRI及SalI酶切pET-24a(+)-H I A，将H II片段连入，之后用SalI及XhoI酶切pET-24a(+)-H I A-H II，将O片段连入，转入大肠杆菌表达菌株BL21(DE3)pLysS，得到包括上述表达基因的表达载体pET-24a(+)-H I A-H II-O。经测序正确后用BamHI及XhoI酶切重组质粒pET-24a(+)-H I A-H II-O，切下片段H I A-H II-O，连入载体PGEX4T-1中，转入大肠杆菌表达菌株BL21(DE3)pLysS，得到包括上述表达基因的表达载体PGEX4T-1-H I A-H II-O。
含有表达载体pET-24a(+)-H I A-H II-O的BL21(DE3)pLysS菌用含100μg/mL硫酸卡那霉素(上海生工生物工程技术服务有限公司，以下简称生工，货号A506636)的500mL LB培养基37℃振荡培养至OD600＝1.0左右，用终浓度为0.25mM的IPTG(生工，货号A100487)28℃诱导6小时。4℃7000rpm离心3分钟收集菌体，每升菌液的菌体用20mL裂解缓冲液(50mM Tirs-HCl，pH8.0，500mM NaCl)重悬，超声破碎，4℃12000g离心20分钟，收集上清过NI柱(BufferA：50mM Tirs-HCl，500mM NaCl pH8.0；BufferB：50mM Tirs-HCl，500mM NaCl，200mM咪唑，pH8.0)。用10倍柱床体积的BufferA平衡Ni-NTA亲和柱之后，加入蛋白样，用10倍介质体积的BufferA洗去未结合的蛋白，之后用25％BufferB洗去杂蛋白，100％BufferB洗脱目的蛋白，此蛋白记为H I A-H II-O-His-tag，作为包被抗原，见图1泳道2，-20℃保存备用。
含有表达载体PGEX4T-1-H I A-H II-O的BL21(DE3)pLysS菌用含100μg/mL氨苄青霉素(生工，货号A100741)的500mL LB培养基37℃振荡培养至OD600＝1.0左右，用终浓度为0.5mM的IPTG(生工，货号A100487)37℃诱导4小时。4℃7000rpm离心3分钟收集菌体，每升菌液的菌体用20mL 裂解缓冲液(50mM Tirs-HCl，500mM NaCl，pH8.0)重悬，超声破碎，4℃12000g离心20分钟，收集上清过GST柱(BufferA：20mM PB+150mM NaCl，Ph7.4；BufferB：25mM Tirs-HCl+10mMGSH，pH8.0)。用10倍柱床体积的BufferA平衡Ni-NTA亲和柱之后，加入蛋白样，用10倍介质体积的BufferA洗去未结合的蛋白，之后用10％BufferB洗去杂蛋白，100％BufferB洗脱目的蛋白，此蛋白记为GST-H I A-H II-O，作为标记抗原，见图1泳道3，-20℃保存备用。
对包被抗原H I A-H II-O-His-tag以及标记抗原GST-H I A-H II-O进行SDS-PAGE电泳，得到图1，其中，泳道1为marker，泳道2为包被抗原，泳道3为标记抗原。由图1可以看出，H I A-H II-O-His-tag以及GST-H I A-H II-O的大小正确，纯度均为90％以上。
实施例2、抗人HIV-1P24杂交瘤细胞株的建立及其单克隆抗体的制备
1.重组P24抗原免疫小鼠
将P24重组抗原(P24-Ag，菲鹏生物股份有限公司)稀释到1.0mg/mL，与弗氏完全佐剂(Sigma-Aldrich公司，货号：F5881)等体积混合，并充分乳化，得到油状乳液。将该乳液以0.2mL的剂量皮下施给BALB/c小鼠(广东省医学实验动物中心：广东省佛山市南海黄岐鄱阳路119号，6周龄雌性，5只)背部位点。第一次免疫14天后腹腔增强免疫，即等量抗原与弗氏不完全佐剂(Sigma-Aldrich公司，F5506)等体积混合，增强免疫到四针后，采尾血，分离血清，用间接ELISA法测定效价，效价高于1∶10000即可用于融合。
融合前3天，用相同剂量抗原与等体积0.9％氯化钠注射液混合腹腔注射追加免疫，免疫方法同上。
2.杂交瘤细胞系的制备
(1)饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈 处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×105个/mL，加入96孔板，150μL/孔，37℃，5％CO2培养过夜。
(2)免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
(3)骨髓瘤细胞的制备
小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)经8-氮鸟嘌呤筛选后，培养至对数生长期，取两大瓶制成细胞悬液，离心，弃上清，用RPMI1640基础培养液重悬，如些重复三次，计数。
(4)细胞融合及HAT选择杂交瘤
将骨髓瘤细胞与免疫脾细胞按1∶10比例混合，在50mL塑胶离心管内用RPMI 1640基础培养液洗1次，1200rpm，离心8分钟。弃上清，将细胞混匀，缓慢加入1mL 50％的PEG1500融合，融合1分钟后加入15mL的RPMI1640基础培养液终止细胞融合。1000rpm，离心5分钟。弃上清，用50mL的RPMI 1640筛选培养液轻轻混悬，平分于10块铺有饲养细胞的96孔板，50μL/孔，37℃，5％CO2培养。培养至第六天，换HT培养液(含HT的RPMI1640完全培养液)两次。
(5)抗体的检测
用0.06M pH9.6碳酸缓冲溶液稀释P24-Ag蛋白使其终浓度为2μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2PBS，0.15mL/孔，37℃封闭2小时，用于检测。上述杂交瘤细胞重组融合后第七天，取细胞上清稀释不同倍数后取0.1mL于上述96孔检测板中，37℃30分钟，PBST洗五次后加入2000 倍稀释的辣根过氧化酶标记的羊抗鼠IgG(羊抗鼠IgG-HRP，菲鹏生物股份有限公司)，37℃30分钟同上洗后，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值。RPMI 1640完全培养液作为阴性对照，共检测有杂交瘤细胞的258孔，最终获得29株稳定分泌P24抗体的细胞株。细胞培养上清效价2.54×103以上。
取其中的三株杂交瘤细胞保藏分别命名为杂交瘤细胞株P24-2F4、杂交瘤细胞株P24-5C2和杂交瘤细胞株P24-3B9。
杂交瘤细胞株P24-2F4于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016211，分类命名：杂交瘤细胞株P24-2F4。
杂交瘤细胞株P24-5C2于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016212，分类命名：杂交瘤细胞株P24-5C2。
杂交瘤细胞株P24-3B9于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016213，分类命名：杂交瘤细胞株P24-3B9。
杂交瘤细胞株P24-2F4分泌的单克隆抗体记为第一抗P24单克隆抗体，杂交瘤细胞株P24-5C2分泌的单克隆抗体记为第二抗P24单克隆抗体，杂交瘤细胞株P24-3B9分泌的单克隆抗体记为第三抗P24单克隆抗体。
3.单克隆抗体的制备
选健壮的BALB/c小鼠(广东省医学实验动物中心：广东省佛山市南海黄岐鄱阳路119号，6周龄雌性)，每只小鼠腹腔注射0.5mL的降植烷；10天后分别腹腔注射1×106个P24-2F4杂交瘤细胞、1×106个P24-5C2杂交瘤细胞和1×106个P24-3B9杂交瘤细胞。接种细胞7～10天后可产生腹水，密切观察动物的健康状况与腹水征象，待腹水尽可能多，而小鼠频于死亡之前，处死小鼠，用滴管将腹水吸入试管中，一般一只小鼠可获5～10mL腹水。收集 腹水，离心取上清，用3倍体积的PBS稀释后滤纸过滤。将所得的滤液在1mL/min的流速下加到一个已用PBS平衡的蛋白G亲和层析柱(GE公司)。然后用PBS以1mL/min的流速洗涤未被蛋白G吸附的物质直至在OD280nm下的吸附值达到基线为止。再用0.1M的甘氨酸洗脱液(pH2.5)洗脱并回收该抗体。所回收的抗体立即用1.5M Tris(pH8.8)中和，跑SDS-PAGE胶，见图2，其中，泳道1为marker，泳道2～4为纯化后的抗P24单克隆抗体。由图2可以看出，三株抗P24单克隆抗体的纯度在95％以上。
上述可分泌抗P24杂交瘤腹水抗体效价均在2.91×106以上。
4.单克隆抗体表位鉴定
用0.06M pH9.6碳酸缓冲溶液稀释纯化好的待鉴定单抗使其终浓度为1μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2PBS，0.15mL/孔，37℃封闭2小时，加入2000倍稀释的辣根过氧化酶标记的P24表位鉴定抗原(P24-Ag2、P24-Ag3、P24-Ag4、P24-Ag5，菲鹏生物股份有限公司)，37℃30分钟，PBST洗5次，拍干，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值，根据反应区分表位，筛选不同表位抗体进行混合优化摸索。
实施例3、HIV检测试剂盒的制备及调试
1.HRP标记抗原的制备
采用NaIO4氧化法。称取8mg辣根过氧化物酶(HRP，英国Biozyme laboratories公司，货号：HRP4)溶解于0.4mL超纯水中，再缓慢滴加0.4mL超纯水新鲜配制的20mg/mL NaIO4溶液，室温下避光轻柔搅拌40分钟后加入48μL乙二醇(取8μL乙二醇溶于40μL蒸馏水中)溶液，室温下避光搅拌40分钟。然后立即加入事先对20mM，pH9.51碳酸盐缓冲液透析2小时，2mg/mL的实施例1得到的纯化好的标记抗原GST-H I A-H II-O 1mL，4℃避光对 20mM，pH9.51碳酸盐缓冲液透析过夜。次日，向混合物中滴加80μL新鲜配制的5mg/mL NaBH4溶液，混匀，4℃静置2小时。将上述液装入透析袋中，对PBS缓冲液(150mM，pH7.4)透析，4℃过夜。加入酶保护剂及终浓度50％的甘油混匀后-20℃避光保存备用。标记抗原GST-H I A-H II-O标记HRP之后的缀合物在以下的文字中称为GST-H I A-H II-O-HRP。
2.生物素标记抗P24单克隆抗体
将实施例2得到的第三抗P24单克隆抗体稀释至1mg/mL，用20mM的PB pH7.4透析24h；称取生物素溶于H2O中配成10mM/L；将处理好的生物素加入透析袋内，PB pH7.4透析液透析12h，得到的生物素标记的第三抗P24单克隆抗体加入终浓度15％的NBS和50％的甘油保存，备用。
3.HIV第四代检测方法
对基因工程重组包被抗原、标记抗原，及抗P24单克隆抗体进行有条件的优化筛选后，建立本发明HIV第四代ELISA检测方法。
1)包被：将实施例1得到的包被抗原H I A-H II-O-Histag、第一抗P24单克隆抗体和第二抗P24单克隆抗体按质量比1∶10∶10(0.2μg∶2μg∶2μg)加入50mM pH 9.6的CB中，混匀进行包被，每孔加入100μL，4℃包被过夜(约22h)；
2)封闭：将包被好的板从4℃取出，平衡至室温，洗板2次；每孔加入120μL封闭液，4℃封闭过夜(约20h)或37℃封闭2h；甩干晾干后待用；
3)加样：将生物素标记的第三抗P24单克隆抗体按体积比1∶1000加入生物素稀释液中，混匀。先每孔中加入25μL P24生物素标记试剂，然后分别在相应孔加入待检标本、阴性、阳性对照75μL，置37℃温育60min。
4)洗涤：弃去孔板中液体，用洗板液洗板5次，每次浸泡30秒；
5)加酶：将标记物GST-H I A-H II-O-HRP、亲和素酶(SA-HRP，菲鹏生物股份有限公司)分别按体积比1∶20000、1∶30000加入酶稀释液中，混匀。每孔加入酶工作液100μL，置37℃温育30min；
6)洗涤：弃去孔板中液体，用洗板液洗板5次，每次浸泡30秒；
7)显色：每孔加入显色剂A液和B液各50μL，轻轻振荡混匀，置37℃避光显色30min；
8)终止：每孔加终止液50μL终止液，轻轻振荡混匀；
9)读值：在酶标仪上，于450nm，630nm处读取OD值。
实施例4、实施例3得到的HIV检测试剂盒的灵敏度和特异性测试
1、灵敏度、特异性比较
a)分别选取了知名的国外(公司A)和国内(公司B)各一家HIV第四代检测试剂产品为对照，对HIV三代国家标准盘同时进行了如下比较，其OD值结果如下表1所示。
表1：实施例3得到的HIV检测试剂盒与现有产品的标准盘检测对比
标本编号 实施例3 公司A 公司B
N1 0.017 0.047 0.013
N2 0.018 0.03 0.008
N3 0.018 0.022 0.044
N4 0.007 0.021 0.01
NS 0.007 0.04 0.028
N6 0.017 0.065 0.009
N7 0.016 0.027 0.034
N8 0.014 0.02 0.021
N9 0.015 0.023 0.002
N10 0.005 0.041 0.011
N11 0.019 0.02 0.014
N12 0.011 0.028 0.039
N13 0.003 0.027 0.01
N14 0.009 0.022 0.028
N15 0.031 0.024 0.038
N16 0.025 0.009 0.031
N17 0.018 0.026 0.023
N18 0.013 0.097 0.003
N19 0.014 0.008 0.027
N20 0.026 0.015 0.012
P1 2.97 2.654 2.637
P2 2.974 2.912 2.734
P3 3.095 2.921 2.603
P4 3.022 2.996 3.19
P5 3.075 2.951 2.763
P6 3.215 2.988 2.914
P7 3.318 2.967 2.761
P8 3.235 3.026 3.148
P9 3.208 2.922 2.676
P10 3.31 3.025 2.836
P11 3.114 2.55 2.694
P12 3.124 2.894 3.05
P13 3.295 2.855 2.793
P14 3.176 2.911 2.864
P15 2.658 0.989 2.696
P16 3.264 2.919 3.094
P17 1.799 2.091 2.705
P18 2.228 2.233 2.288
P19 2.108 1.283 1.142
P20 1.538 0.969 0.628
S1 0.048 0.059 0.003
S2 0.781 0.465 0.573
S3 1.241 1.321 1.123
S4 2.288 1.856 1.499
S5 2.723 2.085 1.377
S6 2.859 1.1 1.047
b)为了进一步考查本试剂盒在临床上的表现，选择了用日本Dainabot有限公司生产的免疫层析法试剂盒(Determine HIV-1/2)验证之后的198份阳性以及3000份阴性标本，进行检测，结果如下表2所示。
表2：实施例3得到的HIV检测试剂盒与现有产品的阳性和阴性检测对比
Figure PCTCN2016112933-appb-000001
由表1和表2可以看出，实施例3得到的HIV检测试剂盒与国内外同类产品的灵敏度、特异性均有一定优势。
2、对HIV-O亚型检测的比较
为了验证三种试剂对HIV-O亚型的检测效果，将购自英国国家生物标准与检定所(National Institute for Biological Standards and Control，NIBSC)的HIV-O型标本进行了检测，其结果(OD值)如下表3所示。
表3：实施例3得到的HIV检测试剂盒与现有产品的HIV-O检测对比
  菲鹏 公司A 公司B
HIV-O标本I 1.588 1.248 0.025
HIV-O标本II 1.021 0.988 0.175
HIV-O标本III 0.665 0.142 0.115
由表3可以看出，在对HIV-O标本的检测上，实施例3得到的HIV检测试剂盒比现有知名的国外公司A检测灵敏度略占优势，较国内公司B具有明显优势。
3、对HIV-1P24检测的比较
为了验证三种试剂对HIV-1P24标本的检测效果，用上述三种HIV第四代检测试剂对P24中检所盘进行了检测，检出差异标本，其结果(OD值)如下表4所示。
表4：实施例3得到的HIV检测试剂盒与现有产品的P24中检所盘检测对比
标本编号 菲鹏 公司A 公司B
P5 0.944 0.633 0.406
P6 0.569 0.321 0.434
P10 3.012 0.945 0.569
由表4可以看出，在对P24中检所盘的检测上，实施例3得到的HIV检测试剂盒比现有产品有更高的灵敏度，而且可能防止漏检。
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种HIV重组抗原，其特征在于，包括依次连接的HIV-1亚型段、HIV-2亚型段和HIV-O亚型段；
    所述HIV-O亚型段为由SEQ ID No.3所示的核苷酸序列编码得到的多肽。
  2. 根据权利要求1所述的HIV重组抗原，其特征在于，所述HIV-1亚型段为由SEQ ID No.1所示的核苷酸序列编码得到的多肽，或者所述HIV-1亚型段为由SEQ ID No.4所示的核苷酸序列编码得到的多肽。
  3. 根据权利要求1所述的HIV重组抗原，其特征在于，所述HIV-2亚型段为由SEQ ID No.2所示的核苷酸序列编码得到的多肽。
  4. 一种表达基因，其特征在于，包括依次连接的HIV-1片段、HIV-2片段和HIV-O片段；
    所述HIV-O片段的序列如SEQ ID No.3所示。
  5. 根据权利要求4所述的表达基因，其特征在于，所述HIV-1片段的序列如SEQ ID No.1所示，或者所述HIV-1片段的序列如SEQ ID No.4所示。
  6. 根据权利要求4所述的表达基因，其特征在于，所述HIV-2片段的序列如SEQ ID No.2所示。
  7. 一种表达载体，其特征在于，包括如权利要求4～6中任一项所述的表达基因。
  8. 根据权利要求7所述的基因表达载体，其特征在于，所述表达载体为pET-24a(+)或PGEX4T-1。
  9. 一种HIV检测试剂盒，其特征在于，包括如权利要求1～3中任一项所述的HIV重组抗原、标记物标记的所述HIV重组抗原、第一抗P24单克隆抗体、第二抗P24单克隆抗体和第三抗P24单克隆抗体。
  10. 根据权利要求9所述的HIV检测试剂盒，其特征在于，所述第一抗P24单克隆抗体为杂交瘤细胞株P24-2F4分泌的单克隆抗体，所述杂交瘤细胞株P24-2F4的保藏编号为CCTCC NO：C2016211；
    所述第二抗P24单克隆抗体为杂交瘤细胞株P24-5C2分泌的单克隆抗体， 所述杂交瘤细胞株P24-5C2的保藏编号为CCTCC NO：C2016212；
    所述第三抗P24单克隆抗体为杂交瘤细胞株P24-3B9分泌的单克隆抗体，所述杂交瘤细胞株P24-3B9的保藏编号为CCTCC NO：C2016213。
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