WO2018119868A1 - 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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WO2018119868A1
WO2018119868A1 PCT/CN2016/112994 CN2016112994W WO2018119868A1 WO 2018119868 A1 WO2018119868 A1 WO 2018119868A1 CN 2016112994 W CN2016112994 W CN 2016112994W WO 2018119868 A1 WO2018119868 A1 WO 2018119868A1
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hiv
recombinant antigen
fragment
subtype
segment
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PCT/CN2016/112994
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French (fr)
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李瑞净
孟媛
于秀玲
刘俊鹏
王益琼
林育佳
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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 Human immunodeficiency virus
  • infection is widespread around the world, which is a serious threat to people's health. Therefore, HIV infection must be monitored.
  • the development of simple, accurate and rapid testing products, early detection of HIV infection, and the elimination of AIDS have become the primary means of prevention and control of AIDS.
  • Immunological detection is mainly a means of detecting specific reactions of antigens and antibodies. Because it can amplify and display detection signals using isotopes, enzymes, and chemiluminescent substances, it is often used to detect proteins, hormones, etc. Trace substances.
  • Immunoassay has undergone different periods such as radioimmunoassay, fluorescent immunoassay and enzyme-labeled immunoassay.
  • Chemiluminescence immunoassay is a new stage in the development of immunoassay. Its environmental protection, rapid and accurate characteristics have been widely recognized.
  • Radioimmunoassay was launched in China in the 1970s. It is still widely used in hospitals at or above the county level. The products are in a recession, and they have basically withdrawn from clinical applications internationally, and the product life cycle has ended.
  • Enzyme-linked immunosorbent assay (ELISA), which was launched in China in the 1980s, is now widely used in clinical institutions at all levels, and is the basic method for clinical immunodiagnosis in China. The products are in maturity; Internationally, it was launched in the 1970s and is still in clinical use. The products are in a recession.
  • Chemiluminescence Immunoassay (CLIA) was introduced into China in the 1990s. Individual hospitals are now carrying out individual projects. The products are in the introduction or growth stage. The international rise in the 1980s has been widely used in clinical practice and has become clinical immunization. The pillar method of diagnosis is at maturity, the instrument is highly automated, and the reagent is serialized.
  • Chemiluminescence immunoassay can be divided into three categories according to their different markers, namely direct chemiluminescence immunoassay, chemiluminescent enzyme immunoassay, and electrochemiluminescence immunoassay.
  • enzymatic chemiluminescence mainly includes horseradish peroxidase (HRP) system, alkaline phosphatase (AP) system, and xanthine oxidase system.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • xanthine oxidase system xanthine oxidase system.
  • the common feature of enzymatic luminescence is that the enzyme as a label in the luminescence process is not substantially consumed, and the luminescent agent is excessive in the reaction system, so the luminescence signal is strong and stable, and the luminescence time is long, so the detection method is simple and the cost is low.
  • CLIA chemiluminescent microparticle immunoassay
  • CMIA method is more sensitive: its sensitivity can reach 10 -16 mol / L (RIA is 10 -12 mol / L), and can be detected as a chemiluminescent substrate (such as AMPPD) the ratio of the concentration of alkaline phosphatase chromogenic substrate was 5x10 5-fold more sensitive, and thus to lower serum antibody concentrations of the samples HIV can be detected effectively, reduce false negatives; 2) CMIA having linear kinetics wider Scope: The luminescence intensity is linearly between 4 and 6 orders of magnitude and the concentration of the measured substance, which is superior to the range of the enzyme immunoassay absorbance (OD value) of 2.0, although the RIA is also broad.
  • OD value enzyme immunoassay absorbance
  • long optical signal duration the glow type of CLIA produces optical signals that can last for hours or even a day. Simplified experimental operation and measurement.
  • Long service period The effective period can be more than 1 year. The radioactive immunoassay is only valid for one month due to the decay of radioisotope, and the enzyme-linked substrate has poor storage, which can not be compared with chemiluminescence. Labor efficiency is also conducive to promotion and application.
  • CMIA method eliminates the need of ELISA repeated sampling, washing plate, etc., can effectively reduce the coefficient of variation and improve repeatability; 6) CMIA method uses automatic enzyme immunoassay analyzer, high degree of automation, can be used for a large number of specimens quickly And accurate detection.
  • An HIV recombinant antigen comprising a HIV-2 subtype segment and an HIV-1 subtype segment
  • the HIV-1 subtype segment is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 1;
  • the HIV-2 subtype fragment is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 2.
  • An expression gene comprising a HIV-2 fragment and an HIV-1 fragment ligated in sequence
  • the sequence of the HIV-1 fragment is shown in SEQ ID No. 1;
  • the sequence of the HIV-2 fragment is shown in SEQ ID No. 2.
  • An HIV detection kit comprising the above-described conjugate of HIV recombinant antigen and magnetic microspheres and the HIV recombinant antigen labeled with a marker.
  • the HIV test kit has the specificity, high sensitivity, good repeatability, excellent stability, wide range of measurement, and detection. High degree of automation and no environmental pollution.
  • Figure 1 is a SDS-PAGE electropherogram of the first HIV recombinant antigen and the second HIV recombinant antigen obtained in Example 1.
  • An embodiment of the HIV recombinant antigen comprises a sequentially linked HIV-2 subtype segment and an HIV-1 subtype segment.
  • the HIV-2 subtype fragment is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 2.
  • the HIV-1 subtype is a polypeptide encoded by the nucleotide sequence shown in SEQ ID No. 1.
  • the above HIV recombinant antigen also includes OSMY linked to the end of the HIV-2 subtype segment away from the HIV-1 subtype segment. That is, the above HIV recombinant antigen includes a sequence of OSMY, HIV-2 subtype and HIV-1 subtype which are sequentially linked.
  • Expression genes for expression of the above HIV recombinant antigen include HIV-2 fragments and HIV-1 fragments which are ligated in sequence.
  • the sequence of the HIV-1 fragment is shown in SEQ ID No. 1, and is designated as H I .
  • HIV-2 fragment The sequence of the HIV-2 fragment is shown in SEQ ID No. 2 and is designated as H II.
  • the expression gene for expression of the above-described HIV recombinant antigen further includes a sequence (which can be referred to as an OSMY fragment) for expressing OSMY which is ligated to the end of the HIV-2 fragment away from the HIV-1 fragment.
  • a sequence which can be referred to as an OSMY fragment
  • 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.
  • Expression vectors comprising the above expressed genes are also disclosed.
  • the expression vector comprising the above expressed gene may be pET-24a (+) or pET-21 (+).
  • the expression vector of the above expressed gene has a His-tag at the C-terminus of the multiple cloning site.
  • the expression vector including the above-mentioned expressed gene can be obtained by designing a primer to amplify a target fragment according to the sequence of the above-mentioned expressed gene, and the upstream primer of the OSMY fragment carries a BamHI site,
  • 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 H I fragment carries the SalI site
  • the downstream primer carries the XhoI cleavage site.
  • the PCR fragment was digested with the corresponding restriction endonuclease, and the OSMY fragment was ligated into the expression vector pET-24a(+) digested with BamHI and EcoRI to obtain the recombinant plasmid pET-24a(+)- OSMY, then pET-24a(+)-OSMY was digested with EcoRI and SalI, and the H II fragment was ligated, then pET-24a(+)-OSMY-H II was digested with SalI and XhoI, and the H I fragment was ligated.
  • the plasmid was transformed into E.
  • the coli expression strain BL21(DE3)pLysS to obtain an expression vector pET-24a(+)-OSMY-H II-H I including the above-mentioned expressed gene.
  • the recombinant plasmid pET-24a(+)-OSMY-H II-H I was digested with EcoRI and XhoI, and the fragment H II-H I was excised, ligated into the vector pET-21(+), and transferred into the large intestine.
  • the strain expresses the strain BL21(DE3)pLysS, and the expression vector pET-21(+)-H II-H I including the above expressed gene was obtained.
  • the expression vector pET-21(+)-H II-H I including the above-mentioned expression gene and the expression vector pET-24a(+)-OSMY-H II-H I including the above-mentioned expression gene can be used for the above-mentioned HIV recombinant antigen. expression.
  • 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 present invention discloses an HIV detection kit according to an embodiment, comprising the above-mentioned conjugate of HIV recombinant antigen and magnetic microspheres and the above-mentioned HIV recombinant antigen labeled with a marker.
  • the HIV recombinant antigen and the magnetic microsphere are coupled by a chemical crosslinking method.
  • the magnetic microspheres carry at least one of an amino group and a carboxyl group.
  • the marker is alkaline phosphatase.
  • This HIV test kit is a third-generation HIV test reagent that detects HIV antibodies by the CMIA method.
  • a marker alkaline phosphatase
  • a marker is labeled on the HIV recombinant antigen to obtain a marker-labeled HIV recombinant antigen, and the HIV recombinant antigen is coupled with the magnetic microsphere to obtain a conjugate of the HIV recombinant antigen and the magnetic microsphere.
  • a luminescent substrate for example, 3-(2-helixadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2- Dioxetane (AMPPD) acts to continuously emit visible light and read the optical signal through a photomultiplier tube.
  • the HIV antibody detection kit has the advantages of high specificity, high sensitivity, good repeatability, excellent stability, wide measurement range, high degree of detection automation and no environmental pollution.
  • SEQ ID No. 1 named H I
  • SEQ ID No. 2 designated H II
  • primers were designed to amplify the fragment of interest.
  • the upstream primer of OSMY full-length fragment carries the BamHI site
  • the downstream primer carries 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 H I fragment carries the primer.
  • the SalI site, the downstream primer with XhoI restriction site, the PCR fragment was recovered by restriction endonuclease digestion, and the OSMY fragment was ligated into the expression vector pET-24a(+) after digestion with BamHI and EcoRI.
  • the recombinant plasmid pET-24a(+)-OSMY was obtained, and then pET-24a(+)-OSMY was digested with EcoRI and SalI, and the H II fragment was ligated, and then pET-24a(+) was digested with SalI and XhoI.
  • -OSMY-H II the H I fragment was ligated into E.
  • the coli expression strain BL21(DE3)pLysS and the expression vector pET-24a(+)-OSMY-H II-H I including the above expressed gene was obtained.
  • the recombinant plasmid pET-24a(+)-OSMY-H II-H I was digested with EcoRI and XhoI, and the fragment H II-H I was excised, ligated into the vector pET-21(+), and transferred into the large intestine.
  • the strain expresses the strain BL21(DE3)pLysS, and the expression vector pET-21(+)-H II-H I including the above expressed gene was obtained.
  • BL21(DE3)pLysS strain containing the expression vector pET-21(+)-H II-H I, containing 100 ⁇ g/mL Ampicillin (Shanghai Shenggong Bioengineering Technology Service Co., Ltd., hereinafter referred to as the labor, No. A100741) 500mL LB medium was shaken at 37 °C to an OD600 1.0, with a final concentration of 0.25 mM IPTG (artificial work, item number A100487) ) induction at 28 ° C for 6 hours. The cells were collected by centrifugation at 7000 rpm for 3 minutes at 4 ° C.
  • the cells of each liter of bacteria were suspended in 20 mL of 50 mM Tirs-HCl, pH 8.0, sonicated, centrifuged at 12000 g for 4 minutes at 4 ° C, and the supernatant was collected through a Q sepharose fast flow column (GE).
  • GE Q sepharose fast flow column
  • the target protein in order to improve the purity and antigen specificity, the Q column eluted sample continued to the NI column (BufferA: 50 mM Tirs-HCl, 350 mM NaCl, pH 8.0; Buffer B: 50 mM Tirs-HCl, 350 mM NaCl, 200 mM imidazole pH 8) .0,), after equilibrating the Ni-NTA affinity column with 5 times the bed volume of Buffer A, add the Q column to elute the protein sample, wash away the unbound protein with 5 times the media volume of Buffer A, and then use 10%. BufferB washes away the heteroprotein, 100% BufferB elutes the target protein, the purity is about 90%, and the first HIV recombinant antigen is obtained, and stored at -20 ° C for use.
  • BufferA 50 mM Tirs-HCl, 350 mM NaCl, pH 8.0
  • Buffer B 50 mM Tirs-HCl, 350
  • the cells of each liter of bacteria were resuspended in 20 mL of lysis buffer (50 mM Tirs-HCl, pH 8.0), sonicated, centrifuged at 12000 g for 20 minutes at 4 ° C, and the supernatant was collected; The protein was precipitated by 15% saturated ammonium sulfate in ice bath, and the supernatant was centrifuged to precipitate the target protein with 25% saturated ammonium sulfate.
  • lysis buffer 50 mM Tirs-HCl, pH 8.0
  • the precipitate was dissolved with 20 mm PB PH7.4, and the corresponding volume of 5X loadingbuffer was added and loaded on 8% SDS- PAGE, 105V 4 °C overnight electrophoresis, the next morning degumming, some strips of 5% CuCl 2 staining, according to the results of the staining of the target protein, into a dialysis bag, with 10mM PB + 1 / million SDS electro-eluting, The voltage was 150V for 4h, and the second HIV recombinant antigen was obtained, and the purity was about 90%, and stored at -20 ° C for use.
  • lane 1 is a marker
  • lane 2 is a first HIV recombinant antigen
  • lane 3 is a Two HIV recombinant antigens.
  • the first HIV recombinant antigen and the second HIV recombinant antigen have the correct molecular weight and a purity of 90% or more.
  • Oxidation Aspirate the dialysis AP into a wide-mouth glass bottle, slowly add 43 mg/mL NaIO 4 solution in an equal volume, and place at 4 ° C for 30 min.
  • Termination of oxidation An equal volume of 1% ethylene glycol was added to the NaIO 4 solution in (2), and placed at 4 ° C for 30 min to terminate the oxidation reaction.
  • Antigen cross-linking The second HIV recombinant antigen was added at a mass ratio of 1:1, loaded into a dialysis bag, placed in a 0.025 mol/L, pH 9.0 carbonate buffer, and dialyzed overnight at 4 °C.
  • the coupling of the first HIV recombinant antigen to the magnetic microspheres is carried out by chemical crosslinking, and the steps are as follows:
  • the HIV test kit includes an AP-labeled second HIV recombinant antigen and a conjugate of the first HIV recombinant antigen and magnetic microspheres.
  • the following method can be used: 25 ⁇ L of magnetic particle suspension, 50 ⁇ L of sample dilution, 50 ⁇ L of sample, reaction for 15 min, 50 mM Tris+0.05% Tween 20, pH 7.5 wash 5 Then, 100 ⁇ L of the diluted pET-26b-OSMY-HIIHI-AP label was added, and the reaction was carried out for 15 min, 50 mM Tris + 0.05% Tween 20, pH 7.5, 5 times, and 100 ⁇ L of the luminescent substrate was added for 5 min to read.
  • CLIA HIV antibody test
  • the sensitivity and background of the HIV test kit obtained in Example 2 were normal, the type II activity was slightly higher than the control kit, and the background was lower than the control kit; the control kit was positive for HIV-I.
  • -1 quality control reactivity is more than double that of the HIV test kit obtained in Example 2
  • the positive HIV-I-2 quality control reactivity is slightly lower than the HIV test kit obtained in Example 2, which is controlled by the negative nature. Poor reactivity.
  • Example 2 also detected 122 confirmed HIV-positive specimens, and all of them were detected, indicating that the HIV test kit obtained in Example 2 has high sensitivity.
  • Example 2 The HIV test kit obtained in Example 2 was passed the national standard plate (batch number 20140930), and the specific test data is shown in Table 2.
  • the HIV antibody detection kit of the present invention can be passed through the national standard.
  • the 2024 clinical specimens were tested.
  • the false positive rate of the HIV test kit obtained in Example 2 was 0.1%, which was much lower than the false positive rate of 0.2% to 1% of the existing HIV double antigen sandwich ELISA products.
  • the specificity of the HIV test kit obtained in Example 2 was good.
  • Example 2 The HIV test kit obtained in Example 2 was subjected to a heat-breaking test at 37 ° C for 3 days and 6 days, and the results of the data are shown in Table 3.
  • the stability test data of the HIV test kit obtained in Example 2 fluctuated within 10%, indicating that the stability of the HIV test kit obtained in Example 2 was acceptable.

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Abstract

一种可以应用于CMIA法检测HIV的HIV重组抗原，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。该HIV重组抗原包括依次连接的HIV-2亚型段和HIV-1亚型段；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；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。

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：
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。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免疫学检验、核酸检测技术等。每种检测技术各有优缺，免疫学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。
免疫学检测主要是利用抗原和抗体的特异性反应进行检测的一种手段，由于其可以利用同位素、酶、化学发光物质等对检测信号进行放大和显示，因此常被用于检测蛋白质、激素等微量物质。
免疫分析经历了放射免疫检验、荧光免疫检验、酶标免疫检验等不同时期，化学发光免疫检验是免疫分析发展的一个新阶段，它环保、快速、准确的特点已得到人们的普遍认识。
放射免疫法(RIA)在中国兴起于20世纪70年代，现仍普遍使用于县级以上医院，产品处于衰退期，在国际上现已基本退出临床应用，产品生命周期已终结。酶联免疫法(ELISA)，在中国兴起于20世纪80年代，现普遍使用于各级临床机构，为我国临床免疫诊断的基本方法，产品处于成熟期； 国际上兴起于20世纪70年代，现仍在临床应用，产品处于衰退期。化学发光免疫法(CLIA)20世纪90年代导入中国，现个别较大医院开展个别项目，产品处于导入期或成长期；国际上兴起于20世纪80年代，现已被临床普遍使用，成为临床免疫诊断的支柱方法产品处于成熟期，仪器自动化程度高，试剂系列化状态。
化学发光免疫分析根据其标记物的不同可分为三大类，即直接化学发光免疫分析、化学发光酶免疫分析、电化学发光免疫分析法。其中酶促化学发光主要包括辣根过氧化物酶(HRP)系统、碱性磷酸酶(AP)系统、黄嘌呤氧化酶系统等。酶促发光的共同特点为发光过程中作为标记物的酶基本不被消耗，而反应体系中发光剂过量，因此发光信号强而稳定，且发光时间较长，故检测方式简单、成本较低。
目前，对于HIV的检测方法较多，其中最常用的有ELISA法、胶体金，核酸检测等，随着医疗技术的发展，化学发光微粒子免疫分析法(CMIA)开始用于HIV抗体的检测。相较于ELISA法，1)CMIA法的灵敏度更高：其灵敏度可达10-16mol/L(RIA为10-12mol/L)，又如化学发光底物(如AMPPD)可检测出的碱性磷酸酶的浓度比显色底物要灵敏5x105倍，因而对于血清中HIV抗体浓度较低的标本也可有效检出，减少假阴性的发生；2)CMIA具有更宽的线性动力学范围：发光强度在4～6个量级之间与测定物质浓度间呈线性关系，这与显色的酶免疫分析吸光度(OD值)为2.0的范围相比，优势明显，虽然RIA也有较宽的线性动力学范围，但放射性限制了其应用；3)光信号持续时间长：辉光型(glow type)的CLIA产生的光信号持续时间可达数小时甚至一天。简化了实验操作及测量。4)使用期长：有效期可长达1年以上，放射免疫分析由于放射性同位素的衰变，一般有效期只有一个月，而酶联的底物贮存性差，都无法与化学发光相比，有效期长可以提高劳动效率，也利于推广应用。5)CMIA法免去了ELISA反复加样、洗板等操作，可有效降低变异系数，提高可重复性；6)CMIA法采用全自动酶免疫分析仪，自动化程度高，可用于大量标本的快速、准确检测。
然而，目前市场上并没有特异性和灵敏度较好的采用CMIA法的HIV检测试剂盒出现。
发明内容：
基于此，有必要提供一种灵敏度较好的可以应用于CMIA法检测HIV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-2亚型段和HIV-1亚型段；
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；
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。
一种表达基因，包括依次连接的HIV-2片段和HIV-1片段；
所述HIV-1片段的序列如SEQ ID No.1所示；
所述HIV-2片段的序列如SEQ ID No.2所示。
一种HIV检测试剂盒，包括上述的HIV重组抗原与磁微球的偶联物以及标记物标记的所述HIV重组抗原。
这种HIV重组抗原应用于HIV检测试剂盒后，通过CMIA法检测HIV抗体，经过试验验证，HIV检测试剂盒具有特异性强、灵敏度高、重复性好、稳定性优异、可测定范围宽、检测自动化程度高和无环境污染等优点。
附图说明
图1为实施例1中得到的第一HIV重组抗原以及第二HIV重组抗原的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-2亚型段和HIV-1亚型段。
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-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。
上述HIV重组抗原还包括连接在HIV-2亚型段远离HIV-1亚型段的一端的OSMY。即，上述HIV重组抗原包括依次连接的OSMY、HIV-2亚型段和HIV-1亚型段。
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-2片段和HIV-1片段。
HIV-1片段的序列如SEQ ID No.1所示，记为H I。
HIV-2片段的序列如SEQ ID No.2所示，记为H II。
用于表达上述HIV重组抗原的表达基因还包括连接在HIV-2片段远离HIV-1片段的一端的用于表达OSMY的序列(可记为OSMY片段)。
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。
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pET-21(+)。
具体的，上述表达基因的表达载体多克隆位点C端有His-tag。
包括上述表达基因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，OSMY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，H I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，OSMY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(+)-OSMY，之后用EcoRI及SalI酶切pET-24a(+)-OSMY，将H II片段连入，之后用SalI及XhoI酶切pET-24a(+)-OSMY-H II，将H I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(+)-OSMY-H II-H I。经测序正确后用EcoRI及XhoI酶切重组质粒pET-24a(+)-OSMY-H II-H I，切下片段H II-H I，连入载体pET-21(+)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pET-21(+)-H II-H I。
包括上述表达基因的表达载体pET-21(+)-H II-H I以及包括上述表达基因的表达载体pET-24a(+)-OSMY-H II-H I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重组抗原与磁微球的偶联物以及标记物标记的上述HIV重组抗原。
优选的，HIV重组抗原与磁微球的偶联物中，HIV重组抗原与磁微球通过化学交联法偶联。
优选的，HIV重组抗原与磁微球的偶联物中，磁微球带有氨基和羧基中至少一种。
优选的，标记物标记的HIV重组抗原中，标记物为碱性磷酸酶。
这种HIV检测试剂盒为第三代HIV检测试剂，通过CMIA法检测HIV抗体。将标记物(碱性磷酸酶)标记在HIV重组抗原上得到标记物标记的HIV重组抗原，将HIV重组抗原与磁微球偶联得到HIV重组抗原与磁微球的偶联物。HIV重组抗原与磁微球的偶联物、样本中HIV抗体以及标记物标记 的HIV重组抗原形成的复合物，可以与发光底物(例如，3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1，2-二氧环乙烷(AMPPD))作用，持续发出可见光，通过光电倍增管读取光信号。
经过试验验证，这种HIV抗体检测试剂盒具有特异性强、灵敏度高、重复性好、稳定性优异、可测定范围宽、检测自动化程度高和无环境污染等优点。
以下为具体实施例。
实施例中采用试剂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、HIV重组抗原的制备
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，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)，设计引物扩增目的片段。OSMY全长片段上游引物带有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，H I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，OSMY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(+)-OSMY，之后用EcoRI及SalI酶切pET-24a(+)-OSMY，将H II片段连入，之后用SalI及XhoI酶切pET-24a(+)-OSMY-H II，将H I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(+)-OSMY-H II-H I。经测序正确后用EcoRI及XhoI酶切重组质粒pET-24a(+)-OSMY-H II-H I，切下片段H II-H I，连入载体pET-21(+)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pET-21(+)-H II-H I。
含有表达载体pET-21(+)-H II-H I的BL21(DE3)pLysS菌，用含100μg/mL 氨苄青霉素(上海生工生物工程技术服务有限公司，以下简称生工，货号A100741)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 50mM Tirs-HCl，pH8.0悬浮，超声破碎，4℃12000g离心20分钟，收集上清过Q sepharose fast flow柱(GE)(BufferA：50mM Tirs-HCl，pH8.0；BufferB：50mM Tirs-HCl+1MNaCl，pH8.0)。用5倍柱床体积的Buffer A平衡Q柱之后，加入裂解上清液，用5倍介质体积的Buffer A洗去未结合的蛋白，之后用10％BufferB洗去杂蛋白，35％BufferB洗脱目的蛋白；为了提高纯度及抗原特异性，Q柱洗脱后的样品继续过NI柱(BufferA：50mM Tirs-HCl，350mM NaCl，pH8.0；BufferB：50mM Tirs-HCl，350mM NaCl，200mM咪唑pH8.0，)，用5倍柱床体积的Buffer A平衡Ni-NTA亲和柱之后，加入Q柱洗脱蛋白样，用5倍介质体积的Buffer A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，纯度达90％左右，得到第一HIV重组抗原，-20℃保存备用。
含有表达载体pET-24a(+)-OSMY-H II-H I的BL21(DE3)pLysS菌用含100μg/mL硫酸卡那霉素(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0.5mM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，pH8.0)重悬，超声破碎，4℃12000g离心20分钟，收集上清；用15％饱和硫酸铵冰浴沉淀杂蛋白，离心取上清用25％饱和硫酸铵冰浴沉淀目的蛋白，离心，沉淀用20mmPB PH7.4溶解，加入相应体积的5Xloadingbuffer后上样于8％SDS-PAGE，105V 4℃过夜电泳，第二天早上拆胶，取部分胶条5％CuCl2染色，根据染色结果切下目的蛋白，装入透析袋，用10mM PB+1/万SDS电洗脱，电压150V 4h，收样，得到第二HIV重组抗原，纯度90％左右，-20℃保存备用。
对第一HIV重组抗原以及第二HIV重组抗原进行SDS-PAGE电泳，得到图1，其中，泳道1为marker，泳道2为第一HIV重组抗原，泳道3为第 二HIV重组抗原。由图1可以看出，第一HIV重组抗原以及第二HIV重组抗原的分子量大小正确，纯度为90％以上。
实施例2、HIV测试剂盒(CMIA)的制备
1、AP标记第二HIV重组抗原的制备(采用采用NaIO4氧化法)
1)酶透析：配制10mg/mL的AP溶液，纯水透析4℃过夜。
2)氧化：吸出透析好的AP至广口玻璃瓶中，等体积缓慢滴加43mg/mL NaIO4溶液，4℃下放置30min。
3)终止氧化：加入与(2)中NaIO4溶液等体积的1％乙二醇，4℃下放置30min，终止氧化反应。
4)抗原交联：按质量比1∶1加入第二HIV重组抗原，装入透析袋，置0.025mol/L，pH9.0碳酸盐缓冲液中，4℃透析过夜。
5)还原：取出透析袋中液体，按12.5∶1体积比加入10mg/mLNaBH4，4℃2h。
6)提纯：等体积加饱和(NH4)2SO4(pH7.6)溶液沉淀结合物，置4℃2h后，4000r/min离心30min，弃去上清。
7)将沉淀物溶于0.05M Tris-HCl+150mM NaCl，pH7.4中，装入透析袋，置0.05M Tris-HCl+150mM NaCl，pH7.4中，4℃透析过夜。
8)得到的AP标记第二HIV重组抗原等体积加入甘油，-20℃保存备用。
2、第一HIV重组抗原与磁微球的偶联物的制备
第一HIV重组抗原与磁微球的偶联采用化学交联法进行，步骤如下：
1)第一HIV重组抗原(1mg/mL×0.5mL)透析至活化缓冲液(50mM MES pH5.5)；
2)取磁微球(Merk公司，品名：Estapor EM1-100/40)0.1mL(100mg/mL)，用活化缓冲液(50mM MES pH5.5)洗涤4次，每次5mL，最后加1mL活化缓冲液，超声分散；
3)称取3mg EDC(1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐)，用0.3mL 活化缓冲液(预冷)溶解至10mg/mL。立刻在振荡条件下往混合液中加入0.2mL EDC溶液，充分混匀。室温旋转(30rpm)反应0.5～1小时，磁分离，弃去上清；
4)加入透析后的第一HIV重组抗原，用50mM MES pH5.5补足1mL，室温旋转(30rpm)结合30分钟，弃去上清；
5)用洗涤液(50mM Tris-HCl+150mM NaCl+0.05％Tween-20+0.1％ProClin 300，pH7.4)洗2次，每次5mL；加5mL封闭液(洗涤液+1％BSA)，室温旋转(30rpm)反应4小时
6)用洗涤液洗3次，每次5mL；最后加1mL磁珠保存液(50mM Tris+1％BSA)重悬，得到第一HIV重组抗原与磁微球的偶联物，终浓度为10mg/mL固体含量，+2～+8℃保存。
3、检测方法
HIV测试剂盒包括AP标记第二HIV重组抗原以及第一HIV重组抗原与磁微球的偶联物。
这种HIV测试剂盒进行检测时，可以采用如下方法：在反应容器中加入磁微粒悬浮液25μL，样品稀释液50μL，样品50μL，反应15min，50mMTris+0.05％吐温20，pH7.5洗5次，加入100μL稀释后的pET-26b-OSMY-HIIHI-AP标记物，反应15min，50mM Tris+0.05％吐温20，pH7.5洗5次，加入发光底物100μL反应5min，读值。
实施例3、实施例2得到的HIV检测试剂盒的灵敏度和特异性测试
1、灵敏度
a)选取国内一家HIV抗体检测(CMIA)试剂盒为对照，进行了灵敏度测试对比，测试结果如表1所示。
表1：实施例2得到的HIV检测试剂盒与对照试剂盒的灵敏度对比
Figure PCTCN2016112994-appb-000001
Figure PCTCN2016112994-appb-000002
由表1可以看出，实施例2得到的HIV检测试剂盒的灵敏度及本底均正常，II型活性比对照试剂盒稍高，本底比对照试剂盒低；对照试剂盒对阳性HIV-I-1质控的反应性比实施例2得到的HIV检测试剂盒低一倍多，阳性HIV-I-2质控反应性稍低于实施例2得到的HIV检测试剂盒，对阴性质控的反应性较差。
另外，实施例2得到的HIV检测试剂盒还对122份确定的HIV阳性标本进行检测，均检出，说明实施例2得到的HIV检测试剂盒的灵敏度高。
b)实施例2得到的HIV检测试剂盒过国标盘(批号20140930)，具体检测数据如表2。
表2：实施例2得到的HIV检测试剂盒
Figure PCTCN2016112994-appb-000003
Figure PCTCN2016112994-appb-000004
由表2可以看出，本发明HIV抗体检测试剂盒可以过国标盘。
2、临床特异性
对2024份临床标本进行检测，实施例2得到的HIV检测试剂盒的假阳率为0.1％，远低于市场现有HIV双抗原夹心法ELISA产品的假阳率0.2％～1％，说明实施例2得到的HIV检测试剂盒的特异性好。
3、稳定性
对实施例2得到的HIV检测试剂盒进行37℃3天、6天热破实验，数据结果如表3所示。
表3：实施例2得到的HIV检测试剂盒的稳定性试验数据
Figure PCTCN2016112994-appb-000005
Figure PCTCN2016112994-appb-000006
由表3可以看出，实施例2得到的HIV检测试剂盒的稳定性试验数据波动均在10％以内，说明实施例2得到的HIV检测试剂盒的稳定性合格。
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-2亚型段和HIV-1亚型段；
    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；
    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。
  2. 根据权利要求1所述的HIV重组抗原，其特征在于，所述HIV重组抗原还包括连接在所述HIV-2亚型段远离所述HIV-1亚型段的一端的OSMY。
  3. 一种表达基因，其特征在于，包括依次连接的HIV-2片段和HIV-1片段；
    所述HIV-1片段的序列如SEQ ID No.1所示；
    所述HIV-2片段的序列如SEQ ID No.2所示。
  4. 根据权利要求3所述的表达基因，其特征在于，所述表达基因还包括连接在所述HIV-2片段远离所述HIV-1片段的一端的用于表达OSMY的序列。
  5. 一种表达载体，其特征在于，包括如权利要求3或4所述的表达基因。
  6. 根据权利要求5所述的基因表达载体，其特征在于，所述表达载体为pET-24a(+)或pET-21(+)。
  7. 一种HIV检测试剂盒，其特征在于，包括如权利要求1或2所述的HIV重组抗原与磁微球的偶联物以及标记物标记的所述HIV重组抗原。
  8. 根据权利要求7所述的HIV检测试剂盒，其特征在于，所述HIV重组抗原与磁微球的偶联物中，所述HIV重组抗原与磁微球通过化学交联法偶联。
  9. 根据权利要求7所述的HIV检测试剂盒，其特征在于，所述HIV重组抗原与磁微球的偶联物中，磁微球带有氨基和羧基中至少一种。
  10. 根据权利要求7所述的HIV检测试剂盒，其特征在于，所述标记物标记的所述HIV重组抗原中，标记物为碱性磷酸酶。
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