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Method for preparing HIV fusogenic suppressor peptide by a gene engineering recombination technology

A recombination technology and genetic engineering technology, applied in the field of preparation of HIV fusion inhibitory peptides by recombination technology, can solve the problems of high cost, low recovery rate, complicated production process, etc., and achieve the effect of shortening the production process, not easy to lose, and simple production process

Inactive Publication Date: 2005-09-28
SHENZHEN UNIV
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AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for preparing HIV fusion inhibitory peptides by genetic engineering technology in order to overcome the shortcomings of the current production process of HIV fusion inhibitory peptide T-20, low recovery rate, complicated production process and high cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] CKS mut Construction of / HIV Fusion Inhibitory Peptide Gene

[0018] The following experimental methods refer to "Molecular Cloning Experiment Guide" translated by Jin Dongyan et al. (1995, Science Press). The CKS gene was cloned from the E.coli K12 strain by the Microbial Genetic Engineering Laboratory of the School of Life Sciences, Shenzhen University according to the genebank sequence. After sequence analysis, it was consistent with the Kds B gene sequence of the E.coli K12 strain reported by Genebank. Mutate the last Met codon ATG at the C-terminus of CKS to Thr codon ATC by site-directed mutagenesis to obtain CKS mut Gene. Select the 36th amino acid coding region Tyr Thr Ser Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn GluGln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp on HIV-1 gp160 Phe.

[0019] Synthesize the gene in the coding region according to the common codons in eukaryotic cells. CKS mut The 643-678 artificial synt...

Embodiment 2

[0021] CKS mut Construction of Yeast Expression of / HIV Fusion Inhibitory Peptide Gene and Screening of Engineering Yeast

[0022] PCR was used to convert the above CKS mut Add EcoRI to the 5' end of the / HIV fusion inhibitor peptide gene and add a NotI site to the 3', clone it into the pPIC9EcoRI-NotI secretion expression vector of Pichia methanolic yeast, and construct the pPIC9EcoRI-NotI secretion expression vector of methanolic yeast pPIC CKS / HIV (for the method refer to Jin Dongyan et al. Cloning Experiment Guide "1995, Science Press).

[0023] Yeast transformation was performed according to the method of Faber (1994). Take a single colony of GS115 and inoculate it in 2.0ml YEPD, culture overnight at 28-30°C, then transfer to 200ml YEPD for 5 hours, collect by centrifugation at 6000g×5min, suspend the bacteria in 25mM sodium phosphate buffer (pH7.0) containing 25mM DTT ), placed at 30°C for 15 minutes, collected by centrifugation, and washed twice with 200ml of 10mM Tr...

Embodiment 3

[0026] With CKS mut / HIV Fusion Inhibitor Peptide Gene Engineering Yeast Fermentation and Product Purification

[0027] Growth medium (g / L): yeast nitrogen base 13.4, biotin 4×10 -4 , peptone 20, yeast extract 10, pH 6.0, fermentation medium (g / L): yeast nitrogen source base 13.4, producin 4×10 -4 , peptone 20, yeast extract 10, trace element mixture 5ml.

[0028]100ml of growth medium was placed in a 500ml conical flask, and after inoculation, the culture was shaken at 200r / min and 28°C. After the OD600 of the seed liquid reaches 4.0, it is transferred to a 5-liter self-controlled fermenter for fermentation. The fermentation tank adopts fermentation medium, 200-300r / min, cultivated at 28°C, and the pH value is controlled at 6.0 with ammonia water. The growth of the bacteria was monitored by observing the dissolved oxygen, and the dissolved oxygen was controlled between 25%. When the dissolved oxygen rises, add 2% glycerol, stop adding glycerol after about 48 hours of cul...

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Abstract

The present invention relates to one kind of genetic engineering recombining technological process of preparing HIV fusion inhibiting peptide. Through fusion between the gene corresponding to the 36 amino acid coding area of 643-678 sites in artificially synthesized HIV-1 gp160 and the 3' terminal of mutant reformed CMP-3-deoxy-D-manno-octulosonic acid synthetase gene, fusion gene with enterokinase recognizing cleavage site is formed. The fusion gene is then cloned to methylogrophic yeast Pichia expression vector to obtain engineering yeast strain with high efficiency expressed fusion gene. The engineering yeast strain is used in fermentation, and the fermented matter is extracted and chromatographically purified to obtain fusion protein, which is cut with enterokinase and purified to obtain recombinant HIV fusion inhibiting peptide. The recombinant peptide, test shows, possesses HIV-1 resisting bioactivity.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for preparing HIV fusion inhibitory peptides by recombinant technology. Background technique [0002] AIDS (AIDS) is caused by human immunodeficiency virus (HIV) infection. Since the first clinical cases of AIDS were reported in the 1980s, AIDS has become the most devastating disease ever seen by human beings. According to incomplete statistics, nearly 20 million people have died of AIDS so far. [0003] The advent of anti-HIV drugs, especially cocktail therapy, is a great progress in the treatment of AIDS. More than ten kinds of AIDS drugs currently approved for clinical use are mainly divided into four categories: viral reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors and adjuvant therapy drugs. Among them, the first HIV fusion inhibitory peptide Fuzeon (enfuvirtide, also referred to as T-20) was jointly developed by Roche Company and Trime...

Claims

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Application Information

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IPC IPC(8): C12N15/62
Inventor 刘志刚吉坤美高波
Owner SHENZHEN UNIV
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