HBsAg基因的克隆及其在毕赤酵母和葡萄组织中的表达

发布时间：2018-06-25 18:42

本文选题：毕赤酵母 + 乙肝疫苗　；参考：《西北大学》2005年硕士论文

【摘要】：本研究利用PCR扩增出adr亚型乙肝表面抗原(HBsAg)基因,构建了酵母和植物表达载体,通过巴斯德毕赤酵母和葡萄组织对获得的乙肝表面抗原基因进行表达研究,取得了如下进展: 1 通过PCR将本实验室构建的pBI121/HB植物表达载体中的乙肝表面抗原基因扩增出来,构建了克隆载体pUC19HB,通过酶切对克隆载体进行了鉴定和测序,并和NCBI中的基因序列进行Blast,证实所获得的基因序列和登陆号为AY646444.1的乙肝表面抗原基因序列完全一致。结果表明,HBsAg基因已经克隆至pUC19中。为HBsAg基因构建表达载体奠定了基础。 2 将克隆载体pUC19HB和酵母表达载体pPIC3.5K用相同的限制性内切酶切割,回收目的片段,用T4 DNA连接酶将两者连接,筛选得到酵母表达载体,经酶切鉴定HBsAg基因已经正确地连接到pPIC3.5K上。用LiCL法将重组以后的酵母表达载体导入甲醇型酵母(Pichia.pasteoris)菌株GS115中,获得转化子。将转化子进行PCR筛选,得到酵母重组菌株。在以甲醇为唯一碳源的培养基上培养,对重组酵母菌株进行了诱导表达。经过SDS-PAGE,发现在诱导72小时后目的蛋白表达量最大,而且在23kDa和27kDa处均有目的条带,说明Pichiapastoris成功表达了HBsAg蛋白,并进行翻译后加工修饰。对表达的蛋白用ELISA初步定性检测结果为阳性,表明表达的HBsAg具有免疫活性,所获得的HBsAg基因可以用于蛋白表达研究。实验结果为下一步利用该基因转化植物材料提供有力的保证。 3 以植物表达载体pBI121/HB为基础,成功构建了新的植物表达载体pCAMBIA1301/HB。将所构建的植物表达载体转化入农杆菌LBA4404,利用农杆菌LBA4404介导转入葡萄愈伤组织中。 4 利用葡萄(Vitis vinifera Linn.)作为表达HBsAg的受体材料。以葡萄叶片为诱导材料,在附加1mg/LNAA,0.1mg/L6-BANAA的MS培养基中诱
[Abstract]:In this study, adr subtype hepatitis B surface antigen (adr) gene was amplified by PCR, and yeast and plant expression vectors were constructed. The expression of HBsAg gene was studied by Pichia pastoris and grape tissue. The following progress has been made: 1 the hepatitis B surface antigen gene in the plant expression vector pBI 121 / HB constructed in our laboratory was amplified by PCR. The cloned vector pUC19HBB was constructed. The cloned vector was identified and sequenced by restriction endonuclease digestion. It was confirmed that the obtained gene sequence was identical with that of hepatitis B surface antigen gene with landing number AY646444.1. The results showed that the HBsAg gene had been cloned into pUC19. 2. Cloning vector pUC19HB and yeast expression vector pPIC3.5K were digested with the same restriction endonuclease, and the target fragment was recovered. The yeast expression vector was screened by T4 DNA ligase and the HBsAg gene was identified to be correctly ligated to pPIC3.5K by restriction endonuclease digestion. The recombinant yeast expression vector was introduced into Pichia pastoris (Pichia.pasteoris) strain GS115 by LiCL method and the transformants were obtained. The recombinant yeast strain was obtained by PCR screening of transformants. The recombinant yeast strain was induced and expressed on the medium with methanol as the sole carbon source. SDS-PAGE showed that the target protein was the most expressed at 72 hours after induction, and there were target bands at 23kDa and 27kDa, indicating that Pichiapastoris successfully expressed HBsAg protein and modified it after translation. The positive results of Elisa showed that the expressed HBsAg had immune activity and the obtained HBsAg gene could be used for protein expression. The results provide a strong guarantee for the further transformation of plant materials by using the gene. 3 based on plant expression vector pBI121 / HB, a new plant expression vector pCAMBIA1301 / HBwas successfully constructed. The constructed plant expression vector was transformed into Agrobacterium tumefaciens LBA4404 and transferred into grape callus by Agrobacterium tumefaciens LBA4404. As a receptor material for HBsAg expression. Grape leaves were used as inducers, induced in MS medium supplemented with 1 mg / L NAA 0.1 mg / L 6-BANAA.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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