Construction of Human Hepatocyte Growth Factor Gene Recombinant Lentivirus Vectors and Its Expression in MyobIasts
10.3969/j.issn.1006-7299.2015.01.012
- VernacularTitle:人肝细胞生长因子基因重组慢病毒载体的构建及其在成肌干细胞中的表达
- Author:
Weifang TANG
;
Fei LIU
;
Shicai CHEN
;
Donghui CHEN
;
Meng LI
;
Minhui ZHU
;
Hongiiang ZHENG
- Publication Type:Journal Article
- Keywords:
Human hepatocyte growth factor;
Lenti virus;
Laryngeal muscle;
Myoblasts
- From:
Journal of Audiology and Speech Pathology
2015;(1):45-49
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a gene recombinant lentiviral vector pCMV -G -U6 -hHGF and detect its expression in C2C12 myoblast cells .Methods hHGF gene fragments were obtained and purified by RT -PCR method ,and were cloned to pCMV -G&NR -U6 ,then the restructured lentiviral vector was transformed into e . coli DH5 alpha ,the positive colonies were identified by BamHI and Hind Ⅲ enzyme digestion .The selected positive colonies were tested by PCR and sequencing analysis .The expression plasmids and packing plasmids were co -trans_fected into 293 T cells ,and virus titer was observed under the fluorescence microscope .Furthermore ,transfected C2C12 cells with lenti virus ,and the expression of HGF was detected by PCR and WB methods .ResuIts PCR and sequencing analysis showed that the lentiviral vector was constructed correctly and successfully ,the virus titer was above 1 x 109 IU/mL .The results of PCR and WB showed that HGF expression level in the lentiviral vector group was much higher than those of in blank control and negative control groups ,and yet the expression was stable after 72 hours .ConcIusion The lentiviral vector pCMV -G﹠NR -U6 -hHGF has been successfully constructed ,and stable expressed in C2C12 cells .It provides references for experimental study in the fields of the denervated skeletal muscle fibrosis and nerve regeneration treatment .
