Construction and identification of the recombinant retroviral vector to carry out hypoxia-regulated expression of neurotrophin-3
10.7652/jdyxb201602009
- VernacularTitle:缺氧调控表达的 NT-3逆转录病毒载体的构建及鉴定
- Author:
Junfeng ZHANG
;
Lili SHI
;
Li ZHANG
;
Pengbo YANG
;
Jianshui ZHANG
;
Yong LIU
;
Cunfang QI
;
Xi XU
- Publication Type:Journal Article
- Keywords:
retroviral vector;
hypoxia-regulated expression;
neurotrophin-3 (NT-3)
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
2016;(2):190-194
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and identify the recombinant retroviral vector containing five copies of hypoxia responsive elements (5HRE)and neurotrophin-3 (NT-3 ).Methods Using PCR,enzyme digestion and DNA ligase,5HRE and human derived NT-3 were cloned into the retroviral vector plasmid (pLNCX)to construct the recombinant retroviral vector plasmid pLNCX-5HRE-SV40-NT3-IRES-EGFP.The retrovirus RV-5HRE-NT3 was packaged in the PT67 cells,and then it was purified and concentrated by high-speed centrifugation.After infected for 48 h with the concentrated retrovirus,the number of the EGFP positive cells in the NIH 3T3 cells was counted by fluorescence activated cells and sorted to calculate the retrovirus titer.Results The retroviral vector plasmid,pLNCX-5HRE-SV40-NT3-IRES-EGFP,was successfully constructed,and the retrovirus was packaged and defined as RV-5HRE-NT3.After purification and concentration,the retrovirus titer reached 9.1 × 10 6 cfu/mL. Conclusion The recombinant retroviral vector which carried out hypoxia-regulated expression of NT-3 was successfully constructed.It may provide basis for studies on hypoxia-regulated expression of the exogenous genes.
