本文關鍵詞：細粒棘球絳蟲轉Eg95-EgA31融合基因苜蓿疫苗構建、鑒定和表達　出處：《重慶醫(yī)科大學》2009年碩士論文　論文類型：學位論文

  更多相關文章： 細粒棘球絳蟲 轉基因 苜蓿 疫苗 構建 鑒定

【摘要】： 目的 制作細粒棘球蚴BALB/c鼠模型,從細粒棘球絳蟲原頭節(jié)擴增Eg95和EgA31抗原編碼基因,構建Eg95-EgA31融合基因,克隆入植物表達載體pBI121構建重組質粒pBI-Eg95-EgA31,電穿孔轉化根癌農桿菌(Agrobacterium tumefaciens,At),侵染紫花苜蓿(Medicago Sativa)構建轉基因苜蓿疫苗;分析重組質粒pBI-Eg95-EgA31在紫花苜蓿中表達效率,為進一步研究細粒棘球絳蟲轉基因苜蓿疫苗的免疫機制奠定基礎。 方法 以BALB/c鼠作為宿主,用腹腔注射Eg原頭節(jié)的方法制作細粒棘球蚴BALB/c鼠模型。從Eg包囊中分離原頭節(jié),超聲粉碎后抽提總RNA為模板,按GenBank Eg95和EgA31的cDNA序列設計引物并引入相應的內切酶位點,通過RT-PCR擴增獲得Eg95和EgA31抗原編碼基因,DNA序列分析和凝膠電泳鑒定擴增產物。通過基因拼接法(GeneSplicing by Overlap Extension ,Gene SOEing)將Eg95和EgA31編碼基因用疏水甘氨酸接頭(Gly4Ser)3經PCR擴增融合,構建融合基因Eg95-EgA31,定向克隆到植物表達載體pBI121,構建重組質粒pBI-Eg95-EgA31,用電穿孔法將該質粒導入根癌農桿菌LBA4404株,擴增重組根癌農桿菌(Recombinant Agrobacterium tumefaciens,rAt),提取rAt中的質粒進行DNA序列分析、PCR和瓊脂糖凝膠電泳鑒定,用含有Eg95-EgA31融合基因的rAt菌液侵染苜蓿葉片,卡那霉素抗性篩選愈傷組織體胚(somatic embryo formed from resisitant callus)。待體胚培育至成熟轉基因苜蓿植株后,用SDS-PAGE、Western blot、PCR和RT-PCR方法鑒定轉基因苜蓿。 結果 擴增的Eg95和EgA31抗原基因分別與GenBank Eg95和EgA31的cDNA(AF199354和AF067807)ORF相應序列完全相同;Eg95-EgA31融合基因經基因測序發(fā)現其長度為1016bp,與預期的序列( 471bp+45bp+500bp )一致。酶切鑒定顯示本研究成功地將Eg95-EgA31融合基因克隆于植物表達載體pBI121的多克隆位點,構建了能表達目的蛋白的重組質粒pBI-Eg95-EgA31。將用YEB平板篩選的rAt經PCR鑒定分析,證實pBI-Eg95-EgA31已成功轉化根癌農桿菌,DNA序列分析和瓊脂糖凝膠電泳均證實rAt中的基因序列為1016bp。 SDS-PAGE和Westrn blot證實Eg95-EgA31融合基因在苜蓿中得到表達,表達產物分子質量(Mr)約為37.5KDa,表達效率為約占苜蓿葉總蛋白的0.05%,且能被感染細粒棘球蚴的鼠血清特異識別。通過PCR和RT-PCR成功擴增出轉基因苜蓿1016bp Eg95-EgA31融合基因片段。 結論 1.通過RT-PCR成功地擴增出Eg95和EgA31抗原編碼基因。 2.通過基因拼接法成功地擴增出1016bp Eg95-EgA31融合基因。 3.成功構建了細粒棘球絳蟲重組質粒pBI-Eg95-EgA31。 4.細粒棘球絳蟲重組質粒pBI-Eg95-EgA31成功轉化了根癌農桿菌。 5.成功培育了細粒棘球絳蟲轉Eg95-EgA31融合基因苜蓿。 6.細粒棘球絳蟲重組質粒pBI-Eg95-EgA31能在紫花苜蓿中表達,并且表達的Eg95-EgA31融合基因重組蛋白具有特異的抗原性。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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