摘要
构建含有冷休克蛋白RNA结合基序蛋白3(RBM3)基因的慢病毒表达载体,产毒感染ST细胞,以评价重组慢病毒载体提高RBM3蛋白表达的效果.应用分子生物学技术提取仔猪脾脏组织的总RNA,通过反转录方法扩增RBM3基因,将其连入慢病毒载体Plenti6/v5-DEST中,在感受态细胞DH5α中扩增培养,并进行RBM3基因的测序鉴定,将重组的慢病毒质粒转染293T细胞,收获病毒液,感染293T细胞并提取细胞RNA,用逆转录PCR方法检测RBM3 mRNA在感染病毒的293T细胞中的表达,用含有慢病毒空载体pLenti6/V5-DEST的病毒液、含有重组慢病毒载体pLenti6/V5-RBM3的病毒液和无菌去离子水分别感染ST细胞后通过Western blot方法检测RBM3蛋白在感染病毒的ST细胞中的表达.结果表明:构建的慢病毒载体Plenti6/v5-RBM3经测序分析证实基因序列正确.包装后的慢病毒滴度为107 PFU/mL.逆转录PCR方法检测到感染病毒的293T细胞中,RBM3基因在转录水平上有表达,Western blot方法检测到感染病毒的ST细胞中,空载体组与空白对照组相比,RBM3蛋白表达量接近相同(分别为0.312±0.022和0.282±0.028);过表达组与空白对照组相比,RBM3蛋白表达量(0.568±0.045)显著增加(P<0.05),RBM3蛋白在翻译水平有表达.因此,本研究构建的携带RBM3基因的重组慢病毒能够感染ST细胞,并使其RBM3蛋白表达量增加.
The RBM3 gene was amplified by using PCR, and then inserted into the lentiviral vector Plenti6/V5-DEST; pLenti6/V5-RBM3 was cultured and amplified in the DH5α competent cells; and RBM3 gene sequence was identified. The 293 T cells were transfected with pLenti6/V5-RBM3. The lentivirus was harvested to infect 293 T cells and ST cells. RT-PCR assay was used to evaluate the mRNA expression of RBM3 in the infected 293 T cells, and western blot was conducted to evaluate the protein expression of RBM3 in the infected ST cells. The RBM3 gene inserted into the lentivirus vetor was verified by sequencing. The lentiviral titer was 10^7 PFU/mL. The lentiviruses produced by 293 T cells were efficient to infect 293 T cells and ST cells. The mRNA expression of RBM3 in the infected 293 T cells was up-regulated, and the protein expression of RBM3 in the infected ST cells was up-regulated too. The result indicated that recombined pLenti6/V5-RBM3 lentivirus could infect with ST cells and promote the expression of RBM3.
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2014年第4期590-596,共7页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(31272524)
农业部948计划重点项目(2011-G35)
黑龙江省自然科学基金项目(C201103)资助~~
