论文部分内容阅读
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。
OBJECTIVE: To construct a adenovirus vector containing the double gene of secreted human CD40L extracellular domain (shCD40L) and IκB. Methods: The target genes shCD40L, IκB and IRES2 were amplified respectively and ligated to get the IκB-IRES2 and shCD40L gene fragments. The two fragments were introduced into pGEMT-easy vector and subcloned. After sequencing, The two shuttle plasmids were successfully ligated with pshuttle-CMV vector to obtain shuttle proteins pshuttle-CMV-shCD40L and pshuttle-cmv-IκB-IRES2, respectively. The shuttle plasmid was amplified in DH5α competent cells. Recombinant adenovirus vector containing two target genes was obtained by homologous recombination with AdEasy-1-BJ5183. The recombinant adenovirus was transfected into 293 cells by lipofectamine and packaged into infectious co-expressing IκB-IRES2-shCD40L adenovirus particles. PCR method to identify differences in the expression of the adenovirus supernatant virus titers were detected and used to infect cells PK15 and 293 cells to test its safety. Results: The constructed recombinant adenovirus of IκB-IRES2-shCD40L gene was identified by restriction enzyme digestion and PCR. The titer of the primary adenovirus reached 6.561 × 1012pfu / L. The PCR products were extracted from the virus supernatant The specific fragments of shCD40L (600bp) and IKBa (700bp) were added. There was no obvious abnormality in the morphology of PK15 cells infected with adenovirus, but obvious balloon-like changes appeared in 293 cells. Conclusion: Adenovirus vector co-expressing IκBα-IRES2-shCD40L double gene was successfully constructed.