Construction and identification of Rattus norvegicus microtubule-associated protein 4 gene recombinant adenovirus
- VernacularTitle:大鼠微管相关蛋白4重组腺病毒载体的构建及在细胞中的表达鉴定
- Author:
Yadong FANG
;
Xue XU
;
Yongming DANG
;
Ji ZHENG
;
Xilian ZHANG
;
Jiaping ZHANG
;
Yu CHEN
;
Qiong ZHANG
;
Yuesheng HUANG
- Publication Type:Journal Article
- Keywords:
MAP4;
adenovirus;
cDNA;
microtubule associated protein
- From:Journal of Third Military Medical University
2003;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct recombinant adenovirus vector containing Rattus norvegicus microtubule-associated protein 4 gene,and transfect it into the rat cardiac myocytes cultured in vitro.Methods A pair of primers were designed,and full length MAP4 DNA was cloned from rat total mRNA by PCR.The PCR product was double-digested with restriction endonucleases NheⅠ and NocⅠ,and inserted orientationally into pShuttle2.The plasmid of pShuttle2-MAP4 was double-digested with restriction endonucleases NheⅠ and NocⅠ,and inserted BD Adeno-X~(TM) Virul DNA,named pAd2-MAP4.The non-recombinant adenovirus was screened out with PacⅠ, pAd2-MAP4 was linerized with SwaⅠ,and the recombinant virus genome was transfected into HEK293 cell line for packaging and amplification of Ad-MAP4 virus.The recombinant adenovirus was transfected into rat cardiac myocytes and MAP4 was identified by immunohistochemistry.Results The recombinant adenovirus-MAP4 was constructed successfully and the titer was about 2.3?10~(8) pfu/ml.The expression of MAP4 was enhanced at 48 h after the transfection.Conclusion We have successfully constructed a recombinant adenovirus Ad-MAP4 that has enforced the expression of MAP4 in vivo.
