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Gene clone of hepatitis B virus front S1 region multi-epitope antigen and encoding sequence thereof

A hepatitis B virus, multi-epitope technology, applied in antiviral agents, genetic engineering, plant gene improvement, etc., can solve the problems of insufficient sensitivity and stability

Inactive Publication Date: 2007-07-04
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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  • Description
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  • Application Information

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Problems solved by technology

[0005] The methods for anti-PreS1 antibody detection have been reported in related literature [respectively: Theilmann L. et al., Hepatology, 6(2): 186-190 (1986); Alberti A. et al., Hepatology, 12(2): 199- 203(1990)], which is basically an indirect ELISA using artificially synthesized PreS1 peptide as the coated antigen, but it often has deficiencies in sensitivity and stability.

Method used

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  • Gene clone of hepatitis B virus front S1 region multi-epitope antigen and encoding sequence thereof
  • Gene clone of hepatitis B virus front S1 region multi-epitope antigen and encoding sequence thereof
  • Gene clone of hepatitis B virus front S1 region multi-epitope antigen and encoding sequence thereof

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Embodiment Construction

[0035] The specific construction method of the multi-epitope antigen gene clone (pres1me) in the pre-S1 region of hepatitis B virus is as follows:

[0036] 1. Synthesize 9 oligonucleotide fragments required for the pres1me gene (see SEQ ID NO.2, synthesized by Shanghai Shenergy Gambling Biotechnology Co., Ltd.).

[0037] 2. The 9 oligonucleotide fragments are both primers and templates, and the multi-epitope antigen gene in the pre-S1 region of hepatitis B virus is synthesized by PCR reaction. The ExpandTM Long TemplatePCR system is used for PCR synthesis and splicing reaction.

[0038] Synthesis of the pres1me gene (Figure 2)

[0039] 0.5 μl each of oligonucleotide fragments 1-9 (30 μM)

[0040] dNTP (10mM) 2.0μl

[0041] 10×PCR buffer 5.0μl

[0042] DNA polymerase mixture (10 units / μl) 1.0μl

[0043] Sterilized redistilled water 35μl

[0044] The PCR reaction was performed under the following conditions: 35 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 74°C fo...

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Abstract

The invention relates to biotechnology field and discloses the gene cloning preslme and its code sequence PRES1ME of multiple-epitope antigen at hepatitis b virus pres region (1) (PreS1). The preslme includes the B cell epitope section 4 at HBV genome pres region (1), and it is indicated that its reaction result with pronuclear fusion expression product GST-PRES1M of gst gene and pre-S1 antigen of hepatitis bvirus ELISA detection kit. The pre-S1 antigen of hepatitis bvirus can be detected in hepatitis b virus serum after peraration of ELISA plate with pres1me, and the serum from GST-PRES1ME immuned mice can identify four Presl epitope synthetic peptide and pre-S1 full- length protein. So pres1me and its expression product PRES1ME can be used to prepare checking agent of HBV pre-S1 antibody or antigen and hepatitis b vaccine.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a gene clone pres1me of the multi-epitope antigen of the pre-S1 region of hepatitis B virus (PreS1) and its encoded protein PRES1ME and their use in the preparation of hepatitis B vaccine and the pre-S1 antibody of hepatitis B virus or Application in Antigen Diagnosis. Background technique [0002] Hepatitis B Virus (HBV) infection is a serious global health problem. In high-incidence areas such as China, Southeast Asia, and Africa, about 10% of the population is infected or has been infected with HBV. The HBV genome includes four open reading frames (ORFs) that overlap with each other to varying degrees. ORF-S encodes HBsAg major protein, medium protein and large protein, which are encoded by ATG at three different positions in the same reading frame, and thus have the same C-terminus. The large protein starts translation upstream of the start codon of the medium prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12N15/51A61K39/29A61P1/16A61P31/20G01N33/576G01N33/68G01N33/53
Inventor 龚育平
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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