论文标题:猪轮状病毒VP7基因在栗酒裂殖酵母中的表达及鉴定
Expression of Porcine Rotavirus VP7 Gene in Schizosaccharomyces Pombe and Identification
论文作者
论文导师 王丕武,论文学位 硕士,论文专业 作物遗传育种
论文单位 吉林农业大学,点击次数 88,论文页数 59页File Size3811K
2006-06-01论文网 http://www.lw23.com/lunwen_134257027/
Porcine Rotavirus;VP7;Schizosaccharomyces pombe;expression;identification
猪轮状病毒(PRV)属于呼肠孤病毒科轮状病毒属，为无囊膜、双股RNA病毒。该病毒是引起婴幼儿及多种幼龄动物急性腹泻并致死亡的主要病原体。在养猪生产上，A组PRV是引起幼猪腹泻的主要病毒性病原体之一。幼猪感染PRV后，因腹泻使机体免疫力急剧下降，易继发多种细菌性感染，导致幼猪成活率下降，存活者生长缓慢或停滞，给养猪业生产带来巨大经济损失。PRV含有11个节段的基因组，他们编码9种蛋白，即结构蛋白VP1、VP2、VP3、VP4、VP6、VP7和非结构蛋白NS34、NS35及NS28。其中构成病毒外壳主要成分的VP7蛋白，是划分病毒血清型的依据，也是诱导机体产生特异性中和抗体的最主要抗原。因此对该基因的克隆及其在酵母菌中的转化、表达、鉴定，对于进一步研究VP7蛋白在植物中的表达及研制PRV基因工程疫苗都具有重要意义。 本实验以RG-2质粒为模板，根据VP7完整序列设计引物进行PCR扩增，得到1000bp左右的特异片段。将其连接到pMD18-T载体上，通过筛选培养在加IPTG、X-gal和Amp的LB培养基上的白斑，得到重组质粒pMD18-T-VP7，经PCR和双酶切验证了PCR产物已克隆到pMD18-T-VP7重组质粒中。 用Nhe Ⅰ和Bgl Ⅱ酶切经PCR扩增的VP7基因产物，而后用Alkaline Phosphatase(Calf intestine)酶对双酶切产物进行了去磷酸化，防止VP7片段自连；纯化产物后在T_4 DNA连接酶的作用下，将其定向克隆到经Nhe Ⅰ和Bgl Ⅱ酶切后的pESP-2质粒上，构建成含有VP7基因的酵母重组表达载体pESP-2-VP7。以热激、电击和LiAC转化法将重组质粒pESP-2-VP7导入栗酒裂殖酵母SP-Q01中。通过在不含亮氨酸的EMM+VB1培养基筛选，鉴定重组pESP-2-VP7阳性质粒，并用PCR、双酶切和SDS-PAGE电泳方法分别对转入DH5α和SP-Q01中的重组质粒进行了鉴定。PCR检测重组质粒含有目的片段，对目的片段测序，与VP7完整序列对照，相似度为98.8％；双酶切重组质粒得到了VP7大小的小片段和pESP-2质粒大小的大片段；SDS-PAGE电泳亦出现了相应大小61kda左右的融合蛋白。确认了重组质粒中含有VP7序列，且构建的含VP7基因的表达载体在酵母中成功转化和表达。 同时测量了不同诱导时间对融合蛋白表达的影响，证明在1mmol／L的IPTG诱导72小时，产生的融合蛋白浓度已基本达到最大值，达到约270mg／L。诱导时间超过72小时OD值变化不大，为以后酵母罐大量生产提供一些资料。
Porcine Rotavirus(PRV) is a member of Rotavirus of Reoviridae family, a kind of no capsule membrane, double helix RNA virus. This virus is a cause and major pathogens of infant, child and many kinds of young animals died of acute diarrhoea. In pig production, A group PRV virus is one of the main cause and virulence pathogens of diarrhoea. After young pigs infected by PRV, the organisms immunity sharp drop cause by diarrhoea, and easily affect by many kinds of bacterial infections, leading to young pig living rate declined, and survivors grow slowly or stagnation, bring tremendous economic losses to the production of pig fanning industry. PRV containing 11 sections of the genome, its coding nine protein, structural protein VP1, VP2, VP3, VP4, VP6, VP7 and non-structural protein NS34, NS35, NS28. VP7 protein is a major component of the virus envelope, not only a basis to the virus serotype division, but also a main antigen which lure organisms produce idiosyncrasy antibodies. Therefore cloning VP7 genes and the certification of its transformation, expression, identification in Schizosaccharomyces pombe have great significance for further study of VP7 protein expression in plants and the development of nuclear PRV genetic engineering vaccines.This experiment used RG-2 plasmids as templates, according VP7 complete sequence designed the primer, progressing PCR acquired 1000bp length VP7 gene products. Connected it to the pMD18-T vector, through filtration white fleck training in added IPTG, X-gal and Amp LB media, acquired the pMD18-T-VP7 recombinant plasmids, by PCR and double digest identified that the VP7 gene have been cloning in pMD18-T-VP7 successfully.Digest VP7 PCR product with Nhe I and Bgl II, then using Alkaline Phosphatase (Calf intestine) enzyme digest product of digest with double enzyme to phosphoric acid, to prevent VP7 segment from coherence, after purification the product and under the effect of T4 DNA enzymes, directional linking it with the pESP-2 plasmids products of digest with Nhe I and Bgl II, constructions a recombinant plasmids yeast vector pESP-2-VP7. Using heat, electric shocks and LiAC conversion method forced recombinant plasmids pESP-2-VP7 into the S.pombe yeast SP-Q01, basis on weather it can growth in the EMM+VB1 media which without leucine to identify the pESP-2-VP7 positive plasmids, using the PCR, double digest and SDS-PAGE electrophoresis methods to identification the recombinant plasmids in DH5a and SP-Q01. PCR testing proved recombinant plasmids containing purpose size PCR fragments and processing the sequence measurement, comparison with the VP7 complete sequence, degree of similarity is 98.8%; the recombinant plasmids after double enzyme acquired a small segment as the size of VP7 gene and a large segment as the size of pESP-2 plasmids; SDS-PAGE electrophoresis also showed a purpose size of fusion proteins as 61kda. Confirmed that the recombinant plasmids containing VP7 sequences, and the successful Conversion and expression of the recombinant plasmids in S.pombe yeast.
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