本文選題：畢赤酵母 + 乙肝疫苗��； 參考：《西北大學(xué)》2005年碩士論文

【摘要】：本研究利用PCR擴增出adr亞型乙肝表面抗原(HBsAg)基因,構(gòu)建了酵母和植物表達載體,通過巴斯德畢赤酵母和葡萄組織對獲得的乙肝表面抗原基因進行表達研究,取得了如下進展: 1 通過PCR將本實驗室構(gòu)建的pBI121/HB植物表達載體中的乙肝表面抗原基因擴增出來,構(gòu)建了克隆載體pUC19HB,通過酶切對克隆載體進行了鑒定和測序,并和NCBI中的基因序列進行Blast,證實所獲得的基因序列和登陸號為AY646444.1的乙肝表面抗原基因序列完全一致。結(jié)果表明,HBsAg基因已經(jīng)克隆至pUC19中。為HBsAg基因構(gòu)建表達載體奠定了基礎(chǔ)。 2 將克隆載體pUC19HB和酵母表達載體pPIC3.5K用相同的限制性內(nèi)切酶切割,回收目的片段,用T4 DNA連接酶將兩者連接,篩選得到酵母表達載體,經(jīng)酶切鑒定HBsAg基因已經(jīng)正確地連接到pPIC3.5K上。用LiCL法將重組以后的酵母表達載體導(dǎo)入甲醇型酵母(Pichia.pasteoris)菌株GS115中,獲得轉(zhuǎn)化子。將轉(zhuǎn)化子進行PCR篩選,得到酵母重組菌株。在以甲醇為唯一碳源的培養(yǎng)基上培養(yǎng),對重組酵母菌株進行了誘導(dǎo)表達。經(jīng)過SDS-PAGE,發(fā)現(xiàn)在誘導(dǎo)72小時后目的蛋白表達量最大,而且在23kDa和27kDa處均有目的條帶,說明Pichiapastoris成功表達了HBsAg蛋白,并進行翻譯后加工修飾。對表達的蛋白用ELISA初步定性檢測結(jié)果為陽性,表明表達的HBsAg具有免疫活性,所獲得的HBsAg基因可以用于蛋白表達研究。實驗結(jié)果為下一步利用該基因轉(zhuǎn)化植物材料提供有力的保證。 3 以植物表達載體pBI121/HB為基礎(chǔ),成功構(gòu)建了新的植物表達載體pCAMBIA1301/HB。將所構(gòu)建的植物表達載體轉(zhuǎn)化入農(nóng)桿菌LBA4404,利用農(nóng)桿菌LBA4404介導(dǎo)轉(zhuǎn)入葡萄愈傷組織中。 4 利用葡萄(Vitis vinifera Linn.)作為表達HBsAg的受體材料。以葡萄葉片為誘導(dǎo)材料,在附加1mg/LNAA,0.1mg/L6-BANAA的MS培養(yǎng)基中誘
[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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 張艷婷;轉(zhuǎn)HBsAg基因?qū)е聶烟曳淹蛔凅w發(fā)生規(guī)律的研究[D];西北大學(xué);2012年

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本文編號：2067094