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Hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof

A technology for human immunodeficiency and protein mutants, applied in applications, viral peptides, antiviral agents, etc., can solve the problems of low titer of immune protective antibodies and poor cross-reactivity, and achieve enhanced immune response and prokaryotic expression. Increased, strong immunogenic effect

Inactive Publication Date: 2013-05-01
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a candidate antigen for HIV vaccine, natural Tat still has the disadvantages of inducing immune protective antibody titers, poor cross-reactivity, etc.

Method used

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  • Hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof
  • Hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof
  • Hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Obtaining a Tat protein that lacks a hydrophobic region through the construction of a recombinant plasmid and prokaryotic expression

[0047] 1.1. In vitro synthesis of the target gene

[0048] The tat1-101-HCVC122-191 DNA fragment was synthesized by the method of Overlap PCR, and the PCR product was recovered by the gel recovery kit, digested by Ase I and BamH I two restriction endonucleases, and the target fragment was recovered, and then mixed with the same The pPEPTIDE (purchased from TAKARA company) vector after Ase I and BamH I digestion ( figure 2 ) connection, and the connection product was transformed into E.coli Top10 competent cells. The plasmid of the transformed colony of the single clone was extracted and sequenced, and the recombinant plasmid with correct sequencing was named pPEP-Tat1-101-HCVC(122-191).

[0049]DNA fragments encoding Tat1-31 and Tat46-101-HCVC (122-191) peptides were synthesized in vitro by PCR method. The PCR reaction sys...

Embodiment 2

[0060] Example 2: The Tat protein gold-labeled immune complex was obtained by preparation and labeling, and its quality was identified.

[0061] 2.1. Preparation of colloidal gold

[0062] Heat 100ml of 0.01% chloroauric acid solution to boiling, accurately add 0.7ml of 1% trisodium citrate under stirring at 200rpm, and the golden yellow chloroauric acid solution turns purple. Continue heating and stirring for 15 minutes, and then dilute to 100ml after cooling.

[0063] 2.2. Electron microscope detection of colloidal gold particle size and uniformity

[0064] The size and uniformity of colloidal gold particles were detected by transmission electron microscopy, and it was found that the average particle diameter of colloidal gold was 75nm, and the uniformity was good ( Figure 7 ).

[0065] 2.3. Tat△(31-45)-PET protein-labeled colloidal gold solution

[0066]The purified Tat(△31-45)-PET protein was dialyzed against 0.005Mol / L pH 9.0NaCl solution at 4°C for 9 hours to determ...

Embodiment 3

[0071] Example 3: Detection of changes in the immunogenicity of Tat mutants and the impact of colloidal gold labeling on its epitope display through animal immunity tests

[0072] 3.1. Animal immunization experiments

[0073] Take 100 μl of purified full-length Tat-PET, Tat(△31-45)-PET fusion protein and Tat(△31-45)-PET protein gold standard (both protein content is 0.01mg) and Freund’s After mixing equal volumes of the complete adjuvant, subcutaneously inject immunized healthy female six-week-old C57BL mice (Experimental Animal Center, Second Military Medical University) into the paws and abdomen, with 5 experimental mice in each group; after that, a booster immunization is performed every 2 weeks. Incomplete Freund's adjuvant was used for immunization, and a total of 3 booster immunizations were performed, and the method and dose were the same as those of the initial immunization. At the same time, colloidal gold and normal saline control groups were set up. Two weeks afte...

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Abstract

The invention relates to the technical field of biomedical engineering, in particular to a hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof. An HIV type I Tat protein mutant is a hydrophobic-region-deletion Tat delta (31-45) mutant, and the amino acid sequence of the mutant is as shown in SEQ ID NO:1. The invention also provides the applications of the Tat protein mutant in preparation of HIV immunogens, preventative and therapeutic HIV vaccines and HIV detection kits. The Tat delta (31-45) mutant protein and gold-labeling particles thereof are conducive to the stabilization of protein conformation, the enhancement of immunogenicity and the show of protein N-terminal and the 46th-61th amino acid epitopes and are expected to be as candidate antigen of HIV vaccine development and HIV infection detection diagnosis.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a human immunodeficiency virus type I (HIV-1) Tat protein mutant sequence lacking a hydrophobic region, and its use in the preparation of HIV immunogens and in the preparation of preventive and therapeutic HIV Application in vaccines and in the preparation of HIV detection kits. Background technique [0002] Human immunodeficiency virus (Human immunodeficiency virus, HIV) is mainly transmitted through blood, sexual contact, and vertical channels from mother to child, and can cause acquired immunodeficiency syndrome (AIDS), that is, AIDS. According to the statistics of UNAIDS in 2012, there are about 34 million HIV-infected people in the world, and there are about 380,000 HIV-infected people and AIDS patients in my country. AIDS has become a major public health problem facing my country and the world. Although the current use of highly active antiretroviral therapy...

Claims

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Application Information

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IPC IPC(8): C07K14/16A61K39/21A61P31/18G01N33/68C12N15/49C12N15/70C12Q1/70C12Q1/68C12R1/93
Inventor 曹洁杨界潘卫张华群王锦红廖文婷祁培培陈秋莉丁超何婷丁莹莹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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