Preparation method of HPV16aE7/huhsp70 fusion protein

A technology for fusing proteins and proteins, applied in the fields of botanical equipment and methods, biochemical equipment and methods, DNA preparation, etc., can solve the problems of incorrect folding of foreign proteins, low yield of inclusion bodies, etc. good effect

Inactive Publication Date: 2014-10-15
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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AI Technical Summary

Problems solved by technology

[0002] Escherichia coli expression system has the characteristics of fast growth, clear genetic background, high expression level and low cost. It is an ideal expression system for exogenous proteins. Active inclusion body forms exist, and the yield of inclusion body refolding is usually low

Method used

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Embodiment Construction

[0014] A preparation method of HPV16aE7 / huhsp70 fusion protein, comprising the following steps:

[0015] 1. Optimization and transformation of HPV16mE7 gene and gene cloning

[0016] Increase E7-specific MHC-I and MHC-II antigen epitopes on both sides of the E7 gene, pRb binding sites (Cys24Gly, Glu26Gly) in the E7 gene, and CKII phosphorylation sites related to transformation (Ser31Gly, Ser32Gly) ) and zinc finger binding region (Cys58Gly, Cys91Gly) mutations, the optimized and modified E7 gene (mE7) was obtained by gene synthesis, which can not only enhance the cellular immune and humoral immune response but also eliminate its transforming activity.

[0017] 2. Construction of pET30a-mE7, pET30a-huhsp70, pET30a-mE7 / huhsp70

[0018] The mE7 gene was synthesized by the gene synthesis method of Shanghai Jierui Biotechnology Co., Ltd., using the upstream primer P1 and downstream primer P2 of the mE7 gene, the mE7 gene was amplified by PCR and cloned into pMD18T, and then subclo...

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Abstract

The invention discloses a preparation method of HPV16aE7/huhsp70 fusion protein. According to the preparation method, pRb binding sites Cys24Gly and Glu26Gly, CK II sites Ser31Gly and Ser32Gly as well as zinc finger binding domains Cys58Gly and Cys91Gly of wild type E7(wE7) protein are mutated, meanwhile, CTL epitope 11-20aa and 49-57aa as well as Th accessory cell epitope 30-67aa with E7 specificity are added at protein N end and C end of the protein E7 respectively, transformed and optimized HPV16 mE7 gene is obtained, a pET-30a-mE7 prokaryotic expression vector is built, a pET-30a-huhsp70 prokaryotic expression vector of huhsp70 is built, mE7 and huhsp70 are fused, the prokaryotic expression vectors are inserted, prokaryotic expression plasmid pET30a-mE7/huhsp70 is built, protein induction expression is performed in escherichia coli BL21(DE3), expression activities of three kinds of protein are identified with SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blotting, and then soluble analysis and purification of recombinant protein are performed. The preparation method has the advantages of simplicity, feasibility, high universality and good tumor treatment effect.

Description

technical field [0001] The invention relates to a preparation method of HPV16aE7 / huhsp70 fusion protein. Background technique [0002] Escherichia coli expression system has the characteristics of fast growth, clear genetic background, high expression level and low cost. It is an ideal expression system for exogenous proteins. Active inclusion body forms exist, and the yield of inclusion body refolding is usually low. Finding a simple, easy, and versatile method to improve or prevent the formation of inclusion bodies is a difficulty and a bottleneck in the current E. coli expression system. Contents of the invention [0003] The present invention mainly solves the technical problems existing in the prior art, thereby providing a simple, easy-to-operate, highly versatile, and effective preparation method of the HPV16aE7 / huhsp70 fusion protein with good tumor treatment effect. [0004] The above-mentioned technical problems of the present invention are mainly solved by the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/37C12N15/10
Inventor 宗金宝王昌媛孙桂荣
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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