摘要
目的构建并鉴定针对LRIG3基因的小干扰RNA(LRIG3-siRNA)腺病毒表达载体,为膀胱癌基因治疗的研究提供有效的实验工具。方法设计可形成小发夹结构的LRIG3-siRNA模板cDNA序列,克隆至缺陷型腺病毒质粒pSilencer-U6neo,构建LRIG3-siRNA表达质粒pSilencer-LRIG3。鉴定正确后,将pSilencer-LRIG3转染至HEK293细胞,包装成复制缺陷型腺病毒pAd-LRIG3。大量扩增pAd-LRIG3,氯化铯梯度离心纯化,测病毒滴度。pAd-LRIG3感染人膀胱癌细胞T24,RT-PCR法检测LRIG3 mRNA的表达。结果酶切分析、测序鉴定表明pSilencer-LRIG3构建成功,PCR分析表明pAd-LRIG3含有LRIG3-siRNA序列。pAd-LRIG3可抑制LRIG3 mRNA的表达。结论含有LRIG3-siRNA的重组腺病毒构建成功,且具有抑制LRIG3基因mRNA表达的功能。
Objective To construct and identify adenovirus vector that expresses small interfering RNA（siRNA）against the LRIG3 gene, which will enable development of a gene therapy protocol for the treatment of human bladder carcinoma. Methods A LRIG3 siRNA template DNA sequence, capable of forming a small hairpin structure, was designed. After renaturation, it was cloned into the defective adenoviral expression vector pSileneer-U6neo to construct the LRIG3-siRNA expression vector pSilencer-LRIG3. After verification,the pSilencer-LRIG3 vector was co-transfected into HEK 293 cells where they were packed as the replication deficient adenovirus pAd-LRIG3, pad LRIG3 was abundantly amplified and then virus titer was evaluated. The pAd- LRIG3 was used to infect the human bladder cancer T24 cells. RT-PCR was used to detect the LRIG3 mRNA expression in T24 cells. Results Restriction endonuclease and sequencing analyses showed that pSilencer LRIG3 was constructed successfully. PCR analysis indicated that the pAd LRIG3 contained the LRIG3 siRNA sequence. The expression of LRIG3 mRNA was greatly inhibited after transfection in T24 cells. Conclusion The recombinant adenovirus vector containing the LRIG3-siRNA gene was successfully constructed and could specifically inhibit the LRIG3 expression.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2010年第1期18-21,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
湖北省自然科学基金资助项目(No.2004ABA205)
作者简介
袁晓奕，男，1974年生，博士研究生，主治医师
通讯作者，Email：wmyang@tjh．tjmuedu．cn
