【作者】 刘伟；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 硕士

【摘要】 分子伴侣(Molecular chaperones)是一类在进化上非常保守的蛋白质家族,同时也是协助细胞在正常和胁迫条件下保持细胞内稳态的重要蛋白组分,参与许多正常的细胞生理反应过程,对蛋白质功能的发挥具有重要意义。由于在进化过程中,病毒在自然选择压力的作用下,为了保持自身基因组足够小,许多真核、原核宿主的病毒可以直接利用宿主细胞中的分子伴侣或者借助宿主细胞编码病毒所需要的分子伴侣以及一些功能蛋白,来完成病毒增殖的许多相关过程。有研究表明,分子伴侣Jiv90在猪瘟病毒(CSFV)的感染过程中起着重要的作用,这种作用可能是通过Jiv90参与调节CSFV NS2-3蛋白的切割来实现的。为了进一步研究分子伴侣Jiv90在CSFV感染过程中对病毒增殖的调节作用,本研究构建了Jiv90基因真核表达载体和抑制Jiv90基因表达的shRNA干扰载体,为进一步研究分子伴侣Jiv90基因的功能奠定基础,期望为猪瘟的防控提供新的思路。本研究获得了以下结果:(1)根据Jiv90基因序列和真核表达载体pEGFP-C1设计引物,经RT-PCR扩增,成功克隆得到猪Jiv90基因,回收后与pEGFP-C1载体连接,构建重组真核表达载体pEGFP-C1-Jiv90,通过酶切鉴定和测序验证所插入基因片段的正确性。经序列比对发现猪Jiv90基因与牛Jiv90基因核酸水平同源性为95%,说明该分子伴侣在两个物种间同源性极高,可能具有类似的功能。(2)载体转化感受态细菌进行扩增并提取质粒,微量紫外/可见分光光度计检测质粒的浓度及质量,通过脂质体法将pEGFP-C1-Jiv90转染猪脐静脉血管内皮细胞(SUVEC),倒置荧光显微镜鉴定转染效率,经G418抗性筛选得到阳性克隆。Real-time PCR检测Jiv90基因mRNA水平表达上调程度,结果显示转染组较空质粒转染组Jiv90 mRNA表达量上调约16.35倍。细胞接种CSFV Shimen株72h结果表明表达Jiv90基因的SUVEC株细胞死亡率明显高于对照组。Real-time PCR检测接种CSFV 60h后,SUVEC-Jiv90细胞株CSFV RNA表达量相对于SUVEC组上调约4.26倍。(3)利用软件设计针对Jiv90基因4个不同位点的RNA干扰序列,通过化学合成的方法合成正向和反向的DNA oligo ,将其退火后连接shRNA表达载体pGPU6/GFP/Neo,成功构建4个pGPU6/GFP/Neo-Jiv90 shRNA1、shRNA2、shRNA3、shRNA4干扰载体,并测序鉴定了各重组载体的正确性。使用脂质体转染方法将干扰载体转染SUVEC,同时转染针对看家基因GAPDH的干扰质粒pGPU6/GFP/Neo-GAPDHshRNA作为阳性对照,不针对任何特异基因的质粒pGPU6/GFP/Neo-NC shRNA作为阴性对照。通过G418抗性筛选获得了转染干扰载体的阳性细胞,以及对照组阳性细胞。更多还原

【Abstract】 Molecular chaperones are highly conservative protein family in evolution, which play important roles in some functions of proteins, such as assisting the cells under normal and stressful circumstances as so to maintain cell homeostasis and participating in many physiological effect.The reason of using molecular chaperones in viruses, might be keeping their genomes small enough under the selective pressure.In order to accomplish the related processes of virus multiplication, a lot of viruses parasitized in eukaryotic and prokaryotic cells could directly use molecular chaperones in their host cells, or code the virus protein chaperones or some functional proteins. It is reported that, the molecular chaperone Jiv90 plays an important role in classical swine fever virus (CSFV) infection, which might be involved in the adjustment by the Jiv90 cleavage of CSFV NS2-3 protein.To further study the effect of the molecular chaperone Jiv90 in the infection process of CSFV, we constructed the eukaryotic expression vector pEGFP-C1-Jiv90 and siRNA vector expressing short hairpin RNA (shRNA)sections which inhibit the expression of Jiv90 gene. Results from this study formed some important basis for Jiv90 gene function research and provided some new ideas for future researches on CSF prevention. And the results as follows:(1)Primers were designed according to Jiv90 gene and pEGFP-C1 sequences, DNA fragments after RT-PCR recovered from agrose gel were inserted into pEGFP-C1 plasmid, successfully constructed expression vector pEGFP-C1-Jiv90. Then the recombinant plasmid was identified by restriction enzyme analysis and DNA sequencing. After the sequence alignment, it is showed that both the swine Jiv90 gene and the bovine Jiv90 gene had 95% homology at nucleic acid level, which indicated that the molecular chaperone had high homology between the two species and similar functions.(2)Those plasmids were amplified by transfecting the competent cells, and the concentration and purity of its solution were detected by spectrophotometer. Then the plasmid pEGFP-C1-Jiv90 was transfected into SUVEC by liposome, furthermore, the efficiency of transfection was identified by observing from the inverted fluorescence microscope, and the G418-resistant colonies were isolated and multiplied.After transfecting of pEGFP-C1-Jiv90, the mRNA expression of Jiv90 gene in SUVEC was detected by Real-time PCR, which was showed that, compared to the control groups, the experimental groups increased about 16.35 times, as means the gene expression were significantly enhanced. It is showed that cells infected with CSFV Shimen strain after 72 h, which already expressed Jiv90 gene in SUVEC, had an evidently higher mortality rate than control group.The results of Real-time PCR showed that CSFV RNA in SUVEC-Jiv90 compared to the control groups increased about 4.26 times when cells infected with CSFV shimen strain after 60 h.(3)Through software, RNAi sequences were designed by using 4 different sites of Jiv90 gene, and according to the designed sequences, sense and anti-sence DNA oligo were chemically synthesized. In the end, the plasmid vectors pGPU6/GFP/Neo-Jiv90 shRNA1, shRNA2, shRNA3, shRNA4 were successfully constructed by annealing and cloning into pGPU6/GFP/Neo.Then the recombinant plasmids were identified by restriction enzyme analysis and DNA sequencing. Also, Interfering plasmid from GAPDH was established as positive control and interfering plasmid targeting none genes served as negative control. Then the recombinant interfering plasmids were transfected into SUVEC by liposome and the G418-resistant colonies were isolated.更多还原