细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗构建、鉴定和表达

发布时间：2018-01-14 16:36

本文关键词：细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗构建、鉴定和表达　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的制作细粒棘球蚴BALB/c鼠模型,从细粒棘球绦虫原头节扩增Eg95和EgA31抗原编码基因,构建Eg95-EgA31融合基因,克隆入植物表达载体pBI121构建重组质粒pBI-Eg95-EgA31,电穿孔转化根癌农杆菌(Agrobacterium tumefaciens,At),侵染紫花苜蓿(Medicago Sativa)构建转基因苜蓿疫苗;分析重组质粒pBI-Eg95-EgA31在紫花苜蓿中表达效率,为进一步研究细粒棘球绦虫转基因苜蓿疫苗的免疫机制奠定基础。方法以BALB/c鼠作为宿主,用腹腔注射Eg原头节的方法制作细粒棘球蚴BALB/c鼠模型。从Eg包囊中分离原头节,超声粉碎后抽提总RNA为模板,按GenBank Eg95和EgA31的cDNA序列设计引物并引入相应的内切酶位点,通过RT-PCR扩增获得Eg95和EgA31抗原编码基因,DNA序列分析和凝胶电泳鉴定扩增产物。通过基因拼接法(GeneSplicing by Overlap Extension ,Gene SOEing)将Eg95和EgA31编码基因用疏水甘氨酸接头(Gly4Ser)3经PCR扩增融合,构建融合基因Eg95-EgA31,定向克隆到植物表达载体pBI121,构建重组质粒pBI-Eg95-EgA31,用电穿孔法将该质粒导入根癌农杆菌LBA4404株,扩增重组根癌农杆菌(Recombinant Agrobacterium tumefaciens,rAt),提取rAt中的质粒进行DNA序列分析、PCR和琼脂糖凝胶电泳鉴定,用含有Eg95-EgA31融合基因的rAt菌液侵染苜蓿叶片,卡那霉素抗性筛选愈伤组织体胚(somatic embryo formed from resisitant callus)。待体胚培育至成熟转基因苜蓿植株后,用SDS-PAGE、Western blot、PCR和RT-PCR方法鉴定转基因苜蓿。结果扩增的Eg95和EgA31抗原基因分别与GenBank Eg95和EgA31的cDNA(AF199354和AF067807)ORF相应序列完全相同;Eg95-EgA31融合基因经基因测序发现其长度为1016bp,与预期的序列( 471bp+45bp+500bp )一致。酶切鉴定显示本研究成功地将Eg95-EgA31融合基因克隆于植物表达载体pBI121的多克隆位点,构建了能表达目的蛋白的重组质粒pBI-Eg95-EgA31。将用YEB平板筛选的rAt经PCR鉴定分析,证实pBI-Eg95-EgA31已成功转化根癌农杆菌,DNA序列分析和琼脂糖凝胶电泳均证实rAt中的基因序列为1016bp。 SDS-PAGE和Westrn blot证实Eg95-EgA31融合基因在苜蓿中得到表达,表达产物分子质量(Mr)约为37.5KDa,表达效率为约占苜蓿叶总蛋白的0.05%,且能被感染细粒棘球蚴的鼠血清特异识别。通过PCR和RT-PCR成功扩增出转基因苜蓿1016bp Eg95-EgA31融合基因片段。结论 1.通过RT-PCR成功地扩增出Eg95和EgA31抗原编码基因。 2.通过基因拼接法成功地扩增出1016bp Eg95-EgA31融合基因。 3.成功构建了细粒棘球绦虫重组质粒pBI-Eg95-EgA31。 4.细粒棘球绦虫重组质粒pBI-Eg95-EgA31成功转化了根癌农杆菌。 5.成功培育了细粒棘球绦虫转Eg95-EgA31融合基因苜蓿。 6.细粒棘球绦虫重组质粒pBI-Eg95-EgA31能在紫花苜蓿中表达,并且表达的Eg95-EgA31融合基因重组蛋白具有特异的抗原性。
[Abstract]:Purpose Eg95 and EgA31 antigen coding genes were amplified from Echinococcus granulosus (Echinococcus granulosus) BALB/c mouse model and Eg95-EgA31 fusion gene was constructed. The recombinant plasmid pBI-Eg95-EgA31 was constructed by cloning into plant expression vector pBI121. Electroporation transformation of Agrobacterium tumefaciensus into Agrobacterium tumefaciensus. Transgenic alfalfa vaccine was constructed by infecting Medicago Sativa. The expression efficiency of recombinant plasmid pBI-Eg95-EgA31 in alfalfa was analyzed, which laid a foundation for further study on the immune mechanism of transgenic alfalfa vaccine of Echinococcus granulosus (Echinococcus granulosus). Method The BALB/c mice were used as the host, and the BALB/c mice model was established by intraperitoneal injection of E. granulosus with the method of procephalus, and the proto-cephalic ganglion was isolated from the EG cyst. The total RNA extracted by ultrasound was used as template, and the primers were designed according to the cDNA sequence of GenBank Eg95 and EgA31 and the corresponding endonuclease sites were introduced. Eg95 and EgA31 antigen coding genes were obtained by RT-PCR amplification. The amplified products were identified by DNA sequence analysis and gel electrophoresis. GeneSplicing by Overlap Extension was performed by gene splicing. The Eg95 and EgA31 coding genes were amplified and fused by PCR using the hydrophobic glycine junction Gly4Serf3. The fusion gene Eg95-EgA31 was constructed and cloned into plant expression vector pBI121. the recombinant plasmid pBI-Eg95-EgA31 was constructed. The plasmid was introduced into Agrobacterium tumefaciens LBA4404 strain by electroporation. Recombinant Agrobacterium tumefaciensus rAt. was amplified. The plasmids extracted from rAt were identified by DNA sequence analysis and agarose gel electrophoresis. The leaves of alfalfa were infected with rAt bacteria containing Eg95-EgA31 fusion gene. Screening callus for somatic embryo formed from resisitant of callus with kanamycin resistance. In vitro embryos were cultured to mature transgenic alfalfa plants. The transgenic alfalfa was identified by SDS-PAGEG Western blot PCR and RT-PCR. Results The amplified Eg95 and EgA31 antigenic genes were associated with cDNA(AF199354 and AF067807 of GenBank Eg95 and EgA31, respectively. The corresponding sequences of ORF were identical; The length of Eg95-EgA31 fusion gene was 1016bp by sequencing. And expected sequence (471bp 45bp 500bp). The results of restriction endonuclease digestion showed that the Eg95-EgA31 fusion gene was successfully cloned into the polyclonal site of plant expression vector pBI121. A recombinant plasmid pBI-Eg95-EgA31 was constructed. RAt screened by YEB plate was identified by PCR. It was confirmed that pBI-Eg95-EgA31 had been successfully transformed into Agrobacterium tumefaciens DNA sequence analysis and agarose gel electrophoresis both confirmed that the gene sequence of rAt was 1016 BP. SDS-PAGE and Westrn blot confirmed that the Eg95-EgA31 fusion gene was expressed in alfalfa, and the molecular weight of the expressed product was about 37.5 KDa. The expression efficiency was about 0.05% of the total protein in alfalfa leaves. The fusion gene fragment of 1016bp Eg95-EgA31 of alfalfa was successfully amplified by PCR and RT-PCR. Conclusion 1. Eg95 and EgA31 antigen coding genes were successfully amplified by RT-PCR. 2. 1016 BP Eg95-EgA31 fusion gene was successfully amplified by gene splicing. 3.Recombinant plasmid pBI-Eg95-EgA31 of Echinococcus granulosus was constructed successfully. 4. The recombinant plasmid pBI-Eg95-EgA31 of Echinococcus granulosus was successfully transformed into Agrobacterium tumefaciens. 5. The Eg95-EgA31 fusion gene of Echinococcus granulosus was successfully cultivated. 6. The recombinant plasmid pBI-Eg95-EgA31 of Echinococcus granulosus can be expressed in alfalfa, and the recombinant protein of Eg95-EgA31 fusion gene expressed has specific antigenicity.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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