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

Construction and identification of Rattus norvegicus microtubule-associated protein 4 gene recombinant adenovirus

目的构建大鼠微管相关蛋白4(m icrotubu le-ossoc iated prote in 4,MAP4)重组腺病毒载体,并转染新生大鼠体外培养的心肌细胞进行表达和鉴定,为真核表达及有关实验研究提供基础。方法设计1对引物,应用逆转录多聚酶链式反应(RT-PCR)技术,从大鼠心肌细胞总mRNA中扩增出MAP4 cDNA,并将MAP4 cDNA克隆到腺病毒穿梭质粒pShuttle2中,构建pShuttle2-MAP4表达质粒。将此表达质粒扩增纯化酶切获得含有目的片段MAP4 cDNA的表达盒与Ad-eno-X V iru l DNA重组并线性化后转染HEK293T细胞,包装产生大鼠MAP4重组腺病毒。并且转染新生大鼠原代心肌细胞,应用免疫组化的方法进行表达鉴定。结果扩增所得的MAP4 cDNA片段经酶切鉴定、PCR扩增、DNA测序最终确定为大鼠MAP4基因,所得大鼠MAP4重组腺病毒滴度为2.3×108pfu/m l。转染原代心肌细胞48 h后MAP4表达显著增强。结论为研究MAP4的生物学活性及功能的基因治疗提供了基础。

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 Nhe Ⅰ and Noc Ⅰ , and inserted orientationally into pShuttle2. The plasmid of pShuttle2-MAP4 was double-digested with restriction endonucleases Nhe Ⅰ and Noc Ⅰ, and inserted BD Adeno-X^TM Virul DNA, named pAd2-MAP4. The non-recombinant adenovirus was screened out with Pac Ⅰ , pAd2-MAP4 was linerized with Swa Ⅰ, 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.