共表达IκBα-IRES2-CD40L胞外区双基因的腺病毒载体构建及鉴定

Construction and identification of recombinant adenovirus vector expressing IκBα-IRES2-shCD40L

目的:构建含有分泌型人CD40L胞外区(shCD40L)和IκB的双基因共表达的腺病毒载体。方法:分别扩增出目的基因片段shCD40L、IκB和IRES2,将目的基因进行连接,得到IκB-IRES2和shCD40L两个基因片段,将其分别导入pGEMT-easy载体进行亚克隆扩增,测序正确后,再将连接成功地两个目的基因片段与分别与载体pshuttle-CMV连接获得穿梭质pshuttle-CMV-shCD40L和pshuttle-cmv-IκB-IRES2,将穿梭质粒在DH5α感受态细胞中扩增。再与AdEasy-1-BJ5183菌同源重组,得到含有两个目的基因的腺病毒骨架,脂质体法转染293细胞,包装成具有感染能力的共表达IκB-IRES2-shCD40L腺病毒颗粒。PCR法对腺病毒的上清液中的表达差异进行鉴定,检测病毒滴度,并用其感染细胞PK15和293细胞以检验其安全性。结果:所构建的IκB-IRES2-shCD40L基因的重组腺病毒,经酶切和PCR鉴定正确,原代腺病毒的滴度达到6.561×1012pfu/L,PCR法从提取的病毒上清液中分别扩增出了是shCD40L(600bp)和IKBa(700bp)的特异性片段,感染了腺病毒后的PK15细胞形态未见明显异常,但293细胞出现了明显的气球样变。结论:成功构建了共表达IκBα-IRES2-shCD40L双基因的腺病毒载体。

AIM：To construct adenovirus expressing vector secretory human CD40L extracellular domain （shCD40L） and IκBα.METHODS：The gene of the shCD40L and the secreting signal peptide was amplified with PCR respectively.Then the CD40L and signal peptide was colligated to get shCD40L segment in vitro.The gene which had cuted from PODB7I κBα and IRES2 were amplified with PCR respectively.After colligated successfully,the gene shCD40L,IRES2,IκBα were inserted into pGEMT-easy to amplify.The gene shCD40L was linked with EGFP and the gene IκBα was linked with IRES2.The two pieces of recombinant gene were inseeted into pshuttle-cmv vector.The pshuttle-cmv-IκBα-EGFP plasmid and pshuttle-cmv-shCD40L-IRES2 plasmid were transdcut into AdEasy adenovirus vector.System and acquired the recombinant plasmid pAdvIκBα-IRES2-shCD40L.The pAdvIκBα-IRES2-shCD40L plasmid harboring was constructed by homologous recombination in E.coil AdEasy-1-BJ5183.Then recombinant vector was propagated in 293 cells and obtain the recombination replication-deficient adenovirus AdvIκBα-IRES2-shCD40L.PCR method was used in identification of recombinant adenovirus vector harboring IκBα-IRES2-shCD40L gene.Expressing products in supernatant adenovirus was identified by PCR method.The safety was evaluated by morphology of PK15 and 293 cells after adenovirus infection.RESULTS：The recombinant IκBα-IRES2-shCD40L adenovirus was generated by homologous and identified by PCR methods.The adenovirus titre reached 6.561×10^12 pfu/L.The adenovirus didn’t replicate in PK15 cells.CONCLUSION：The IκBα-IRES2-shCD40L adenovirus is prepared successfully and its biotic safety is confirmed.