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HIV (Human Immunodeficiency Virus) recombinant fusion antigen as well as expression gene and preparation method thereof

A technology of fusion antigen and gene expression, applied in the field of medicine and biology, can solve the problems of specificity and sensitivity limitation, inability to detect HIV-1 and HIV-2 at the same time, difficulty in purification and renaturation, etc.

Active Publication Date: 2014-06-11
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This has limited the application of recombinant HIV gp41 antigen in antibody detection, especially the specificity and sensitivity.
[0013] Due to the high hydrophobicity of gp41 or gp36, recombinantly expressed antigens are generally expressed in the form of inclusion bodies, and inclusion bodies need to be soluble above pH 11.0, which brings great difficulties to subsequent purification and renaturation
And there are many restrictions on the application of recombinant antigens in immune antibody detection
And a single gp41 or gp36 recombinant antigen can only detect HIV-1 or HIV-2 antibodies alone, and cannot detect HIV-1 and HIV-2 types at the same time

Method used

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  • HIV (Human Immunodeficiency Virus) recombinant fusion antigen as well as expression gene and preparation method thereof
  • HIV (Human Immunodeficiency Virus) recombinant fusion antigen as well as expression gene and preparation method thereof
  • HIV (Human Immunodeficiency Virus) recombinant fusion antigen as well as expression gene and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Design of Fusion Antigen

[0037] For many infectious diseases, the corresponding antigenic epitopes on pathogens become the targets for the development of specific diagnostic reagents and vaccines. HIV infection is no exception, and its gp41 protein is an important detection target. gp41 is the transmembrane glycoprotein of HIV-1, which directly mediates the fusion of HIV and cell membrane, and plays an important role in the process of HIV infection of cells. gp41 also has a neutralizing epitope and an immunodominant domain, which can induce neutralizing antibodies and an early and strong antibody response in the body. 在本发明中,通过分析设计,HIV-1gp41选取HIV标准病毒株HXB2病毒株中的优势表位:536aa-684aa,其对应的氨基酸序列为:tltvq arqllsgivq qqnnllraie aqqhllqltv wgikqlqari laverylkdq qllgiwgcsgklicttavpw naswsnksle qiwnhttwme wdreinnyts lihslieesq nqqekneqel leldkwaslwnwfnitnwlw yikl

[0038] Due to the strong hydrophobicity of this sequence, the sequence was modified and designed by bioinformatics techn...

Embodiment 2

[0052] Example 2 Construction of recombinant antigen prokaryotic expression vector

[0053] The target gene was synthesized by chemical synthesis, cloned into the pMD18-T simple Vector vector, and the T vector containing the target gene was obtained (provided by Invitrogen Company), which was digested by BamH I and HindIII at 37°C for 4-5 hours, and the digested product was After purification and recovery, 6 μl of each was ligated with the plasmid pET-28a digested with the same enzyme, and ligated at 22°C for 1 hour. The ligation product was transformed into competent E. coli DH5α strain by heat shock method, spread on LB plates containing 34 μg / ml Kan, and the positive clones were picked up and sent for sequencing after preliminary identification by colony PCR and enzyme digestion. The sequencing results proved the expression vector The constructed sequence is consistent with the designed gene sequence (as shown in SEQ ID NO.4+SEQ ID NO.5+SEQ ID NO.6). The recombinant expres...

Embodiment 3

[0083] The composition of the fusion antigen of HTV of the present invention is basically the same as that of Example 1, the difference is that the linker is GSGGSGG, and its base sequence composition is ggcagcggcggcagcggcggc (SEQ ID NO.7).

[0084] 3.1 The preparation steps (including purification and renaturation) of the HTV fusion antigen described in this implementation are as in Example 2. The purity of the recombinant antigen purified by the affinity chromatography method reaches 90%.

[0085] image 3 It is the expression identification diagram of the recombinant antigen described in this example; lane 1: protein marker; lane 2: lysed precipitate of expressing bacteria; lane 3: supernatant of lysed expressing bacteria; lane 4: control bacteria without induced expression. Figure 4 It is the purified electrophoresis identification diagram of the recombinant antigen in this example, wherein, lane 1 is purified recombinant protein; lane 2: protein marker.

[0086] 3.2 Th...

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Abstract

The invention discloses an HIV (Human Immunodeficiency Virus) recombinant fusion antigen as well as an expression gene and a preparation method thereof. The HIV recombinant fusion antigen is formed by sequentially combining an amino acid shown as SEQ ID NO.1, a connexon and an amino acid shown as SEQ ID NO.3, wherein the connexon is composed of 6-10 hydrophilic amino acids with small molecular weight and polarity. The HIV recombinant fusion antigen is constructed and inducibly expressed in a plasmid by using the expression gene of the HIV recombinant fusion antigen. The HIV recombinant fusion antigen is applied to HIV-1 and HI-1 antibody detection, can be used for detecting HIV-1 and HIV-2 antibodies at the same time, and has very good detection sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and specifically relates to an HIV recombinant fusion antigen and its expression gene and preparation method. Background technique [0002] AIDS (Acquired Immune System Deficiency, AIDS) is a viral disease that seriously endangers human health and is attracting worldwide attention. The main pathogens causing AIDS are human immunodeficiency viruses type I and type II (HIV-1 and HIV-2). Due to the deepening of immunodeficiency, HIV-infected persons develop from asymptomatic carriers to severe immune failure, leading to the development of various fatal diseases and death. According to WHO's 2007 global health report, as of the end of 2007, there were estimated to be 33.2 million HIV-infected people worldwide, and 2.7 million new HIV-infected people worldwide in 2007 alone. In developing countries, due to various reasons such as economy, culture and education, the prevalence of HIV has become mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/569C12R1/19C12R1/93
Inventor 王继华周腊梅唐时幸
Owner GUANGZHOU WONDFO BIOTECH
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