Construction method for prokaryotic secretory expression vector of protein transduction domain-Apoptin (PTD-Apoptin) fusion protein and application of prokaryotic secretory expression vector

Abstract

The invention relates to a construction method for a prokaryotic secretory expression vector of a protein transduction domain-Apoptin (PTD-Apoptin) fusion protein and the application of the prokaryotic secretory expression vector. The construction method comprises the following steps of: connecting a PTD-Apoptin sequence onto a pET22b(+) secretory expression vector to construct the prokaryotic secretory expression vector pET22b(+)-PTD-Apoptin; secreting an expressed interest protein into the periplasmic space of escherichia coli through isopropyl thiogalactoside (IPTG) induction; extracting the protein of the periplasmic space by an osmose process; performing ultrasonication on remaining cellular components; then, separating supernatant and depositing; and performing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Co-incubation is performed on the PTD-Apoptin fusion protein in the periplasmic space and a gastric cancer cell-823, and the inhibitory actionof the fusion protein on the gastric cancer cell-823 is detected by a methyl thiazolyl tetrazolium (MTT) method. By adopting the construction method, the recombinant PTD-Apoptin protein can be secreted into the periplasmic space of escherichia coli and exists in a soluble state. The recombinant PTD-Apoptin protein of secretory expression has biological activity, and the highest inhibition ratio on the gastric cancer cell-823 is 82.95 percent.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to the construction and application of a PTD-Apoptin fusion protein secretion expression system. Background technique [0002] Today, the treatment of tumors is still a difficult problem. Therefore, finding and preparing preparations with strong selectivity and strong action on tumor cells is the current research direction of tumor therapeutic drugs. The vp3 gene (Apoptin) isolated from chicken anemia virus can induce the apoptosis of abnormal cells such as human tumor cells or transformed cells, but has no effect on normal diploid cells. The apoptosis of tumor cells induced by Apoptin does not require the expression of tumor suppressor gene p53, and is not inhibited by the apoptosis factor Bcl-2, showing the application prospect of Apoptin as an anti-tumor agent. [0003] In order for Apoptin to effectively enter tumor cells, it is necessary to construct a drug molecule ...

AI Technical Summary

Problems solved by technology

However, the cytoplasmic expression of prokaryotic expression systems mostly exists in the form of inclusion bodies. Inclusion bodies are denatured proteins, generally inactive, and require renaturation to restore activity. However, the renaturation efficiency of inclusion bodies is low. Less than the requirements, and only a very small number of proteins are folded into the natural state when the inclusion body is refolded, limiting its activity

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