公开(公告)号：US11499163B2

公开(公告)日：2022-11-15

申请号：US16175812

申请日：2018-10-30

Applicant: SUN YAT-SEN UNIVERSITY

Inventor： Qin Deng ,  Xiao Shen ,  Weiwei Su ,  Zhong Wu ,  Wei Peng ,  Yonggang Wang

IPC: C12N15/85 ,  C12N15/64 ,  G01N33/573 ,  C12N9/64

Abstract: The disclosure relates to an expression method of a Haemocoagulase Acutus (Halase) recombinant protein. The method includes the following steps: (1) optimizing a halase gene; (2) performing Polymerase Chain Reaction (PCR) amplification on an optimized halase gene; (3) constructing an Agkis-pMCX expression vector, transforming plasmids to competent cells of an Escherichia coli for amplification, screening in an Amp-resistant manner for positive cloning, sequencing and extracting recombinant plasmids with correct sequencing; (4) transfecting recombinant plasmids to CHO cells; and (5) expressing the recombinant protein and identifying. According to the expression method, a recombinant halase is expressed first using the CHO cells cultured in a serum-free suspension manner; and by utilizing a bioengineering means, the actual production problems of insufficient raw material sources and unstable quality of a snake venom product are solved successfully.

公开(公告)号：US20190048361A1

公开(公告)日：2019-02-14

申请号：US16175812

申请日：2018-10-30

Applicant: SUN YAT-SEN UNIVERSITY

Inventor： Qin Deng ,  Xiao Shen ,  Weiwei Su ,  Zhong Wu ,  Wei Peng ,  Yonggang Wang

IPC: C12N15/85 ,  G01N33/573 ,  C12N15/64

Abstract: The disclosure relates to an expression method of a Haemocoagulase Acutus (Halase) recombinant protein. The method includes the following steps: (1) optimizing a halase gene; (2) performing Polymerase Chain Reaction (PCR) amplification on an optimized halase gene; (3) constructing an Agkis-pMCX expression vector, transforming plasmids to competent cells of an escherichia coli for amplification, screening in an Amp-resistant manner for positive cloning, sequencing and extracting recombinant plasmids with correct sequencing; (4) transfecting recombinant plasmids to CHO cells; and (5) expressing the recombinant protein and identifying. According to the expression method, a recombinant halase is expressed first using the CHO cells cultured in a serum-free suspension manner; and by utilizing a bioengineering means, the actual production problems of insufficient raw material sources and unstable quality of a snake venom product are solved successfully.