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HIV-1 yesisting polypeptide C22, its coding sequence and prepn process

A sequence and encoding technology, applied in the field of genetic engineering, can solve problems such as limiting the efficacy of clinical treatment

Inactive Publication Date: 2006-08-02
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the earlier development is mainly for the enzyme (reverse transcriptase or protease) inhibitors that play a key role in the HIV-1 virus infection process, but due to the emergence of drug-resistant virus variants, the drug needs to be kept stable efficacy, and considering the potential toxic effects of the drug, which greatly limits the efficacy of existing inhibitor series clinical treatments

Method used

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  • HIV-1 yesisting polypeptide C22, its coding sequence and prepn process
  • HIV-1 yesisting polypeptide C22, its coding sequence and prepn process

Examples

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Effect test

Embodiment 1

[0065] Embodiment 1 Preparation of polypeptide C22 clone of the present invention

[0066] 1.1 Obtaining of polypeptide C22 nucleotide chain

[0067] A C22 single-stranded nucleotide template and forward and reverse primers were designed respectively, and a C22 nucleotide chain (SEQID NO: 3) was obtained by PCR reaction.

[0068] The design template and primers are as follows:

[0069] Templet for C22

[0070] 5’GAA TTG GAT AAA TGG GCG TCG CTG TGG AAT TGG TTT AAT ATT ACC AAT TGG CTG TGG TAT

[0071] ATT AAA 3'

[0072] 5'primer for C22 (EcoRI)

[0073] 5'GC GAA TTC GAC GAC GAC GAC AAA GAA TTG GAT AAA TGG GCG 3' (SEQ ID NO: 4).

[0074] 3'primer for C22(XhoI)

[0075] 5' CCG CTC GAG TTA TTT AAT ATA CCA CAG CCA 3' (SEQ ID NO: 5).

[0076] In a 50 μl reaction system (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.0 mM MgCl2, 200 μM dNTPs), 100 pmole of each amplification primer was added. After mixing well, start the PCR cycle: denaturation at 95°C for 1 minute, denaturation at ...

Embodiment 2

[0080] Example 2 Expression and purification of polypeptide C22 of the present invention

[0081] The identified clones were shaken, induced and expressed when the OD value reached 0.5-0.8, induced overnight at 30 degrees Celsius, and collected at 8000g for 10 minutes. Add 20mL of 2% triton in PBS, 20uLDDT, 100uL of lysozyme and PMSF to each 500mL culture medium. Use 500W power to ultrasonically destroy bacteria. Centrifuge at 13000g for 20 minutes after breaking the bacteria, and take the supernatant. Combined with GlutathioneSepharose 4B column, washed with 1×PBS and eluted with reduced glutathione to obtain GST-C22 fusion protein. Such as figure 2 shown. The resulting fusion protein can be digested with protease Enterokinase to obtain polypeptide C22.

Embodiment 3

[0082] Embodiment 3: Physicochemical parameters of polypeptide C22 of the present invention

[0083] According to SDS-PAGE electrophoresis and standard molecular weight curve, the isoelectric point of the polypeptide C22 of the present invention is 6.17. The molecular weight is 2812.22.

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Abstract

The present invention relates to one kind of HIV-1 resisting polypeptide C22, and its expression precursor, coding DNA sequence, monomer polypeptide medicine composite and preparation process. The polypeptide C22 is prepared through expression with fusion protein to obtain the polypeptide precursor, and cleaving the precursor to obtain polypeptide C22. The preparation process is simple, high in final product yield and suitable for industrial production.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention relates to an anti-HIV-1 polypeptide C22 sequence, its coding sequence and its preparation method. Background technique [0002] AIDS (AIDS) is a serious infectious disease that destroys the immune function of the human body after the AIDS virus invades the human body, causing various incurable infections and tumors in the human body, and finally leading to the death of the infected person. [0003] Since the first case of AIDS infection was discovered in China in 1985, AIDS has shown an exponential growth trend in China, and its growth trend has even surpassed that in Africa. Not only the number of infected people has risen sharply, but all the varieties of HIV found in the world have all appeared in China. [0004] The reason why AIDS is rampant all over the world is that the AIDS virus HIV directly invades the human immune system after invading the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/11C12N15/62C12N15/63A61K38/16A61P31/18
Inventor 汪世龙孙晓宇李敏张震
Owner TONGJI UNIV
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