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Method for producing recombinant scorpion toxin protein by adopting silkworm as parasitifer

A technology of scorpion toxin protein and scorpion toxin, which is applied in the fields of baculovirus gene expression system, gene recombination technology, protein purification and activity analysis, can solve the problems of unstable yeast plasmid transformants and high cost of mammalian cells, and achieves Low cost, low production cost, large individual effect

Inactive Publication Date: 2012-04-25
ZHEJIANG UNIV
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  • Claims
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Problems solved by technology

However, yeast plasmid transformants are unstable and expensive to produce in cultured mammalian cells

Method used

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  • Method for producing recombinant scorpion toxin protein by adopting silkworm as parasitifer
  • Method for producing recombinant scorpion toxin protein by adopting silkworm as parasitifer
  • Method for producing recombinant scorpion toxin protein by adopting silkworm as parasitifer

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Embodiment Construction

[0020] 1. Research materials: genetic engineering operation tools, enzymes and PCR amplification kits were purchased from Shanghai Sangon Bioengineering Technology Service Company in my country. Scorpion cDNA was purchased from Clotech Company, TA cloning kit, maps such as figure 1 The shown baculovirus transfer vector pAcGP67b and liposome lipofectin are all products of Invitrogen Company. Products for Ni-NTA affinity chromatography were purchased from Wakochemical Co., Ltd., Japan. DNA sequencing kits were purchased from PE Applied Biosystems. Fetal calf serum FCS, medium TC-100 for insect cell line Sf21 and medium 199 for culturing umbilical vein endothelial cells HUVEC are all products of GibcoBRL. The medium ESF921 of the Fall Armyworm cell line Tn-5 was purchased from Expression systems. Umbilical vein endothelial cells HUVEC, SCS and ECGS are all products of Technoclone GmbH. Spodoptera frugiperda cells Sf21 were cultured at 27°C in TC-100 medium supplemented with ...

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Abstract

The invention discloses a method for producing recombinant scorpion toxin protein by adopting a silkworm as a parasitifer, which mainly comprises the following steps: adopting a scorpion cDNA as a template, adopting 5'-ACGTAGATCTATGGATGGATATATAAG-3' containing BglII enzyme cutting site as a forward primer and adopting 5'-ACGTAGATCT TTACTTTTTTCCAC-3' containing the BglII enzyme cutting site as a reverse primer to carry out PCR gene amplification reaction in 32 cycles to obtain a scorpion toxin gene; digesting pCR2.1-scorpion toxin by restriction endonuclease BglII to obtain a scorpion toxin gene fragment; cloning the scorpion toxin gene fragment in a baculovirus transfer vector pAcGP67b to obtain a recombinant pAcGP67b-scorpion toxin gene, carrying out homologous recombination with baculovirus BmNPV DNA of a silkworm in an insect cell by cotransfection to produce a recombinant virus; expressing in a silkworm body and purifying the recombinant scorpion toxin. Most of the recombinant scorpion toxin protein expressed by a silkworm larva is retained in a fat body of the silkworm; the scorpion toxin is purified from the fat body by an affinity chromatography; and the recombinant scorpion toxin has obvious biological toxicity, is suitable for the requirement of a biopharmaceutical industrialization level and has wide application prospect in the field of future medical and clinic treatment.

Description

technical field [0001] The invention relates to gene recombination technology, baculovirus gene expression system, protein purification and activity analysis, in particular to a method for producing recombinant scorpion toxin protein using silkworm as a host. Background technique [0002] Scorpion toxin protein BmK is a small molecular weight protein synthesized and secreted by scorpions. The gene length is 198 nucleotides, encoding 65 amino acids, pI is 6.3, and the predicted molecular weight is about 8KDa, which has strong toxicity. Scorpion toxin has anti-viral, anti-bacterial, anti-tumor, anti-inflammatory, dredging collaterals, dispelling rheumatism, antispasmodic, tranquilizing, pain-relieving and other functions. Therefore, scorpion toxin has good application value and shows a good application prospect. However, the price of scorpion toxin protein on the market is relatively expensive. Although it can be produced in Escherichia coli by genetic engineering, the scorpi...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C12P21/02C12R1/93
Inventor 相兴伟于少芳吴小锋
Owner ZHEJIANG UNIV
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