【摘要】： 目的:構建日本血吸蟲Sj14FABP和Sj26GST膜錨定共表達質粒pIRES-Sj26-Sj14,并檢測其在體外的表達。 方法:利用RT-PCR法,以日本血吸蟲成蟲總RNA為模板,擴增獲得Sj14FABP與Sj26GST的全長基因。并先將其克隆到真核表達質粒pVAC中,分別獲得重組質粒pVAC-Sj14、pVAC-Sj26。然后分別以pVAC-Sj14、pVAC-Sj26質粒為模板,采用PCR技術擴增出含人白介素2(IL-2 )23個氨基酸的信號肽與人胎盤堿性磷酸酶(PLAP)COOH-末端32個氨基酸的膜錨定序列在內(nèi)的Sj14、Sj26修飾基因。并將該兩個修飾基因共同構建到真核表達載體pIRES上獲得共表達質粒pIRES-Sj26-Sj14,將重組質粒轉染Hela細胞,通過RT-PCR的方法及間接免疫熒光技術檢測Sj26, Sj14基因的膜錨定表達。 結果:經(jīng)過酶切鑒定、PCR、測序證實所克隆的Sj14FABP、Sj26GST基因與報道結果完全一致,重組真核表達質粒pVAC-Sj14、pVAC-Sj26、pIRES-Sj26-Sj14構建成功。并且pIRES-Sj26-Sj14質粒在體外轉染Hela細胞后可表達膜錨定蛋白Sj14與Sj26。 結論:成功構建了日本血吸蟲Sj14FABP和Sj26GST膜錨定雙表達質粒,該質粒轉染人子宮頸癌Hela細胞后可正常表達這兩種蛋白,為下步對其免疫原性免疫反應性及免疫保護作用的進一步的研究奠定了基礎,也給血吸蟲疫苗的研究拓寬了思路。
[Abstract]:Aim: to construct the co-expression plasmid pIRES-Sj26-Sj14, of Schistosoma japonicum Sj14FABP and Sj26GST and to detect its expression in vitro. Methods: the full-length genes of Sj14FABP and Sj26GST were amplified by using total RNA of adult Schistosoma japonicum as template by RT-PCR method. The recombinant plasmid pVAC-Sj14,pVAC-Sj26. was obtained by cloning it into eukaryotic expression plasmid pVAC. Then the pVAC-Sj14,pVAC-Sj26 plasmids were used as templates, The Sj14,Sj26 modified genes including the signal peptide containing 23 amino acids of human interleukin 2 (IL-2) and the membrane anchoring sequence of 32 amino acids of (PLAP) COOH- terminal of human placental alkaline phosphatase (PLAP) COOH-) were amplified by PCR. The two modified genes were co-constructed into eukaryotic expression vector pIRES to obtain the co-expression plasmid pIRES-Sj26-Sj14,. The recombinant plasmid was transfected into Hela cells. The membrane anchoring expression of Sj26, Sj14 gene was detected by RT-PCR and indirect immunofluorescence technique. Results: PCR, sequencing confirmed that the cloned Sj14FABP,Sj26GST gene was in good agreement with the reported results. The recombinant eukaryotic expression plasmid pVAC-Sj14,pVAC-Sj26,pIRES-Sj26-Sj14 was successfully constructed. Moreover, pIRES-Sj26-Sj14 plasmid can express membrane anchoring protein Sj14 and Sj26. after transfection of Hela cells in vitro. Conclusion: the double expression plasmids of Schistosoma japonicum Sj14FABP and Sj26GST membrane anchoring have been constructed successfully. These two proteins can be expressed normally after transfected into human cervical cancer Hela cells, which is the next step to identify the immunogenicity of Schistosoma japonicum and Schistosoma japonicum. The further study of immunoreactivity and immune protection has laid a foundation for the research of Schistosoma japonicum vaccine.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

【參考文獻】

相關期刊論文 前10條

1 李柳哲,石佑恩,江侃,寧長修;不同載體的日本血吸蟲DNA疫苗誘導小鼠免疫效果的觀察[J];華中科技大學學報(醫(yī)學版);2003年01期

2 劉述先;血吸蟲疫苗研制的策略、現(xiàn)狀及展望[J];中國血吸蟲病防治雜志;1994年S1期

3 李柳哲,石佑恩;日本血吸蟲表膜蛋白23kDa的DNA疫苗的研制及其誘導小鼠的保護性免疫[J];中國血吸蟲病防治雜志;1999年02期

4 李柳哲,石佑恩,寧長修,鄧偉文,韓家俊;日本血吸蟲混合DNA疫苗免疫效果觀察[J];中國血吸蟲病防治雜志;2001年04期

5 馮新港,余新炳,吳忠道,周俊梅;日本血吸蟲SjFABPc重組質粒裸DNA免疫小鼠的研究[J];中國血吸蟲病防治雜志;2001年05期

6 胡雪梅,張兆松,吳�，|,李春林,蘇川,季e鹲，

本文編號：2429747