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Surface glucoprotein gp160 of recombination expression human acquired immunity defact virus 1

A surface glycoprotein and expression cassette technology, applied in the field of bioengineering, can solve problems such as high or low expression levels

Inactive Publication Date: 2006-01-11
上海安久生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, gp160 expressed in this way has deficiencies in the integrity of antigenicity, the level of expression, and the rapid isolation of gp160 after expression.

Method used

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  • Surface glucoprotein gp160 of recombination expression human acquired immunity defact virus 1
  • Surface glucoprotein gp160 of recombination expression human acquired immunity defact virus 1
  • Surface glucoprotein gp160 of recombination expression human acquired immunity defact virus 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Obtaining HIV-1 gp160 gene encoded by 6 histidines at the C-terminal by PCR method

[0046] The DNA plasmid pN2gpt-gp160 (US Patent No. 5,445,953) carrying the HIV-1 gp160 gene was used as a template, and the following pair of primers were used to carry out conventional DNA polymerase chain reaction (PCR) under the action of polymerase.

[0047] 5' end primer:

[0048] 5'GCT CTA GAG AGC AGA AGA CAG TGG CAA TG 3' (SEQ ID NO: 3)

[0049] 3' end primer:

[0050] 5’CGA ATT CTA TTA GTG GTG ATG GTG ATG GTG TAG CAAAAT CCT TTC CAA GCC CTG 3' (SEQ ID NO: 4, the underlined part corresponds to 6His)

[0051] The resulting amplified product was digested with XbaI and inserted into the working plasmid pUC19 (purchased from New England Biolab) that had been digested with XbaI and blunt-ended at the HidIII site. Enzyme ligation according to conventional methods, transformed into Escherichia coli DH5α, positive clones were selected, and DNA plasmids were prepared. The prepared D...

Embodiment 2

[0054] Mutation of the 516th amino acid arginine encoded on the HIV-1 gp160 gene to serine

[0055] Using the plasmid PAC-gp160 as a template, PCR reactions were performed with the following two pairs of primers, respectively.

[0056] Reaction 1:

[0057] Primer 1: 5' end primer (SEQ ID NO: 3) in Example 1

[0058] Primer 2: 5’GGA ACA AAG CTC CTA TTC CCA CTG C G C TTT TTTCTC TCT GC 3' (SEQ ID NO: 5) (note: the underlined G is T in the wild-type sequence)

[0059] Reaction 2:

[0060] Primer 3: 5'GTG GGA ATA GGA GCT TTG TTC CTT GG 3' (SEQ ID NO: 6).

[0061] Primer 4: 3' end primer (SEQ ID NO: 4) in Example 1.

[0062] The products obtained from PCR reaction 1 and reaction 2 were mixed as a template, and the primers (SEQID NO: 3 and 4) in Example 1 were used to carry out PCR reaction again. The resulting PCR product was digested with XbaI, and inserted into the plasmid pUC19 (purchased from New England Biolab Company) that had been digested with XbaI and blunted at the H...

Embodiment 3

[0066] Preparation of recombinant vaccinia virus with gp-160 gene

[0067] Digest PAC-gp160mu with endonuclease EcoRI, separate the gp-160mu DNA fragment, and insert it into the commonly used expression plasmid PVAC that has been digested by EcoRI (see the plasmid structure map image 3 )middle. The obtained PVAC plasmid carrying the gp-160mu gene was named PVAC-gp160mu.

[0068] Monkey kidney cell line BSC (ATCC CCL-26) was grown in complete DMEM medium to a density of 80%. BSC cells were infected with vaccinia virus New York strain (purchased from American Type Culture Collection, ATCC VR-1536) at a concentration of 0.1 PFU. The infected BSC cells were incubated in a 37°C carbon dioxide incubator for 2 hours. The PVAC-gp160mu plasmid DNA was transfected into BSC cells infected with vaccinia virus. The cells after DNA transfection were cultured in a carbon dioxide incubator at 37 degrees Celsius for 2 days, and then the cells were freeze-thawed and lysed, and then collect...

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Abstract

A process for preparing the modified surface glucoprotein gp160 of human acquired immune deficiency virus 1 in the cowpox virus system, the cowpox virus used to prepare the protein gp160, and the prepared protein gp160 are disclosed. Its advantages are high output and high purity.

Description

technical field [0001] The present invention relates to the field of bioengineering, more specifically to a method for expressing the surface glycoprotein gp160 of human acquired immunodeficiency virus type I (abbreviated as "gp160 protein") in a vaccinia virus system, and also to a carrier for producing the surface glycoprotein and the produced surface glycoprotein gp160. Background technique [0002] Human acquired immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and HIV carriage. AIDS patients will gradually destroy the body's immune system and eventually die. Although HIV carriers are not sick, it has caused a huge blow to their work and life and poses a threat to the health of others. [0003] HIV-1 virus infection can be determined by measuring the body's anti-HIV-1 virus antibody. The HIV-1 virus has multiple antigens. One of the surface glycoproteins was named gp160 because of its molecular weight of 160kd. After gp160 is synthesized in the c...

Claims

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Application Information

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IPC IPC(8): C12N15/863C12N15/48C07K14/16C07K1/14
Inventor 楼觉人
Owner 上海安久生物科技有限公司
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