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Production of specific micro-antibody for oarium cancer

An anti-idiotype and ovarian cancer technology, applied in the field of genetic engineering, can solve the problems of low expression level and unsuitable for large-scale production

Inactive Publication Date: 2006-11-01
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] But since 6B11V L V H The hC gene is a eukaryotic gene, and the currently disclosed 6B11V L V H The G and C content of the starting sequence at the 5′ end of the hC gene is relatively high, and contains more rare codons of E. coli. The expression level in E. coli is very low, so it is not suitable for large-scale production

Method used

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  • Production of specific micro-antibody for oarium cancer
  • Production of specific micro-antibody for oarium cancer

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Experimental program
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Effect test

Embodiment 1

[0066] Obtaining the target gene and constructing the expression plasmid

[0067] Acquisition of the target gene

[0068] The human ovarian cancer anti-idiotypic microantibody gene comes from the Gynecological Oncology Research Center of Beijing People's Hospital. We designed primers to optimize it. The optimized gene sequence is shown in SEQ ID NO:1. A pair of primers were designed, and the unoptimized gene 5' end sequence was modified by PCR method, and NcoI and EcoRI restriction sites were introduced at the same time.

[0069] Construction of expression plasmids

[0070] Escherichia coli JF1125 was selected as the host strain, and the strain was transformed, and the constructed expression plasmid pTrcHisA / 6B11V L V H hC was transformed into Escherichia coli JF1125. Pick single clones (JF1125 / pTrcHisA / 6B11V) from LB plates containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol L V H hC), inoculated into 3ml LB liquid medium, oscillated at 300rpm on a constant tem...

Embodiment 2

[0072] Optimal combination of expression vector and host bacteria

[0073] Escherichia coli JF1125 was selected as the host strain, and the strain was transformed, and the constructed expression plasmid pTrcHisA / 6B11V L V H hC, pBADHisA / 6B11V L V H hC, pBAD / gIIIA / 6B11V L V H hC were transformed into Escherichia coli JF1125 respectively. Single clones were picked from LB plates containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol, inoculated into 3 ml LB liquid medium, shaken at 300 rpm on a constant temperature shaker at 37°C overnight, and inoculated in 20 mL LB (ampicillin Concentration of 100μg / ml and 34μg / ml chloramphenicol) culture medium, cultivated to OD at 37°C, 250rpm 600 =0.5-1.0, the cells are in the logarithmic growth phase at this time; L V H Add IPTG to the culture medium of hC to a final concentration of 1mM, strain JF1125 / JF1125 / pTrcHisA / 6B11V L V H hC and JF1125 / JF1125 / pBAD / gIIIA / 6B11V L V H Add arabinose to the hC culture solution to a fin...

Embodiment 3

[0078]Ovarian cancer anti-idiotypic mini-antibody fermentation

[0079] Prepare 300ml of LB fermentation seed liquid and 2.7L of M9 fermentation medium, and prepare 100ml of 50% glucose and 5% CA feed. Place in a 250ml Erlenmeyer flask. disinfect. Inoculate after cooling the seed solution. After reaching OD 0.6~1, stop the cultivation and inoculate the upper tank. Control the temperature at 37°C, pH = 7.0, cultivate until the OD reaches 3.0, add 0.6g IPTG for induction, and close the tank after 3 hours, during which feeding is fed to maintain the growth of bacteria. The cells were collected by centrifugation, and the results of SDS-PAGE electrophoresis showed that the expression level of the target protein reached 50%.

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Abstract

The invention provides a nucleotide sequence encoding ovarian cancer anti-idiotypic microantibodies, a production method for efficiently producing ovarian cancer anti-idiotypic microantibodies, and related engineering cell construction, expression and purification processes. The optimized ovarian cancer anti-idiotypic microantibody gene is very suitable for expression in Escherichia coli. At the same time, by optimizing the combination of expression plasmids and host bacteria and optimizing the fermentation process, the expression level has been increased, and it has the advantages of high expression and high stability. The invention can obtain pure ovarian cancer anti-idiotypic micro-antibody with high efficiency, convenience and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a method for efficiently producing ovarian cancer anti-idiotypic microantibodies, as well as related engineering cell construction, ovarian cancer anti-idiotypic microantibody expression and purification processes. Background technique [0002] In 1974, Jerne proposed the famous immune network theory based on Burnet's "clonal selection theory" based on modern immunology's understanding of antibody molecular idiotypes. The theory holds that there are idiotypes on any antibody molecules or antigen receptors of lymphocytes, which can be recognized by other lymphocytes in the body to stimulate and induce anti-idiotypes. Based on the mutual recognition of idiotypes and anti-idiotypes, a "network" connection is formed in the immune system, which plays an important role in immune regulation. According to the immune network theory, anti-idiotypic anti...

Claims

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Application Information

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IPC IPC(8): C12N15/13C07K1/14C07K16/18C12N1/21C12N15/63C12N15/70C12P21/02
Inventor 任军黄阳滨孙九如顾小伟赖伟萍沈丽丽
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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