Wistar大鼠热休克蛋白70基因重组腺病毒质粒的构建及病毒制备

目的构建Wistar大鼠热休克蛋白(Heat shock protein 70,HSP70)基因重组腺病毒质粒,并制备重组腺病毒。方法利用RT-PCR技术从Wistar大鼠脾脏组织中扩增HSP70基因序列,并克隆至pMD18-T载体中,测序鉴定后,将克隆的HSP70基因编码阅读框亚克隆至穿梭载体pShuttle-CMV中,并利用Ⅰ-CeuⅠ与Ⅰ-SceⅠ将重组穿梭质粒上的HSP70基因的表达框切下,连接到携带腺病毒骨架载体pAdxsi上。重组腺病毒质粒经PacⅠ酶切线性化后,转染至293(R)细胞进行病毒的包装,获得重组腺病毒pAd-CMV-HSP70,收集病毒上清,感染BRL细胞,Western blot检测BRL细胞中HSP70的表达。病毒颗粒经氯化铯密度梯度离心纯化后,检测重组腺病毒纯度、颗粒滴度及感染性滴度。结果克隆质粒、穿梭质粒经PCR及酶切鉴定,均可见2 269 bp的目的基因条带,测序结果与GenBank登录的序列一致;XhoⅠ酶切结果显示,HSP70基因已正确克隆至腺病毒质粒中;重组腺病毒感染BRL细胞后,细胞中HSP70的表达量显著升高(P<0.01);重组腺病毒纯化后纯度为1.29,颗粒滴度为5.31×1012VP/ml,感染性滴度达1×1011pfu/ml。结论已成功构建Wistar大鼠HSP70基因重组腺病毒质粒,并制备出高滴度的重组腺病毒颗粒,为进一步研究HSP70的生物学活性及作用机制奠定了基础。

Construction of recombinant adenovirus expression vector for Wistar rat heat shock protein 70 gene and preparation of recombinant adenovirus

Objective To construct a recombinant adenovirus expression vector for Wistar rat heat shock protein 70(HSP70) gene and prepare recombinant adenovirus.Methods HSP70 gene was amplified from spleen tissue of Wistar rats by RT-PCR and cloned into vector pMD18-T.The coding reading frame of cloned HSP70 gene was identified by sequencing and subcloned into shuttle plasmid pShuttle-CMV.The constructed recombinant shuttle plasmid pShuttle-CMV-HSP70 was digested withⅠ-CeuⅠandⅠ-SceⅠ,and the obtained expression frame of HSP70 gene was linked to vector pAdxsi carrying adenovirus backbone.The recombinant adenovirus backbone plasmid was linearized by digestion with PacⅠ,and transfected into 293(R)cells for packaging to obtain recombinant adenovirus pAd-CMV-HSP70.BRL cells were infected with the collected virus supernatant,in which the expression of HSP70 was determined by Western blot.Virus particles were purified by cesium chloride density gradient centrifugation,and determined for purity,titer and infectivity.Results Target gene band at a length of about 2 269 bp was observed in the cloning and shuttle plasmids as proved by PCR and restriction analysis,of which the sequence was consistent with that reported in GenBank.Digestion with XhoⅠ showed that HSP70 gene was cloned to adenovirus vector correctly.The expression level of HSP70 in BRL cells infected with recombinant adenovirus increased significantly(P < 0.01).The recombinant adenovirus reached a purity of 1.29 after purification.The titer of virus particles was 5.31 × 1012 VP/ml,while the infectious titer was 1.0 × 1011 pfu/ml.Conclusion The recombinant adenovirus vector for Wistar rat HSP70 gene was constructed successfully,and high titer recombinant adenovirus particles were prepared,which laid a foundation of further study on biological activity and action mechanism of HSP70.