Construction and identification of Rattus norvegicus microtubule-associated protein 4 gene recombinant adenovirus / 第三军医大学学报

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.