本文選題：重組毒素 + 人白介素3�。� 參考：《重慶醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的:構(gòu)建人白細(xì)胞介素(IL-3)與假單胞菌外毒素片段(PE38KDEL)融合基因的原核表達(dá)載體,為進(jìn)一步表達(dá)具有殺傷作用的融合蛋白奠定基礎(chǔ)。 方法: 1)提取人外周血總RNA,經(jīng)RT-PCR擴(kuò)增出IL-3基因,并用二次PCR方法引入柔性連接肽linker片段,PCR產(chǎn)物插入載體PQE30中,轉(zhuǎn)化克隆進(jìn)行酶切鑒定及DNA測(cè)序肯定克隆結(jié)果。 2)以PGEX-4T-1PE38KDEL質(zhì)粒為模板,PCR擴(kuò)增PE38KDEL片段,PCR產(chǎn)物經(jīng)雙酶切后插入到PQE30-IL-3質(zhì)粒中構(gòu)建PQE30-IL-3-PE38KDEL,連接產(chǎn)物轉(zhuǎn)化克隆經(jīng)酶切鑒定及DAN測(cè)序鑒定。 結(jié)果:本研究成功構(gòu)建了原核表達(dá)質(zhì)粒PQE30-IL-3-PE38KDEL.經(jīng)酶切鑒定與預(yù)期結(jié)果吻合,核酸序列測(cè)定顯示插入的IL-3基因及PE38KDEL基因與Genebank報(bào)道的序列完全一致。
[Abstract]:Aim: to construct the prokaryotic expression vector of the fusion gene of human interleukin 3 (IL 3) and pseudomonas exotoxin fragment PE38 KDEL, and to lay a foundation for the further expression of the fusion protein with killing effect. Methods: 1) Human peripheral blood total RNAs were extracted, and IL-3 gene was amplified by RT-PCR, and then inserted into the vector PQE30 by the second PCR method. The transformed clones were identified by restriction endonuclease digestion and the results were confirmed by DNA sequencing. 2) PGEX-4T-1PE38KDEL plasmid was used as template to amplify the PE38KDEL fragment. The product was digested by double enzyme and inserted into PQE30-IL-3 plasmid to construct PQE30-IL-3-PE38KDEL. The ligated product was identified by restriction endonuclease digestion and DAN sequencing. Results: the prokaryotic expression plasmid PQE30-IL-3-PE38 KDEL was successfully constructed. The nucleic acid sequence analysis showed that the inserted IL-3 gene and PE38KDEL gene were identical with the sequence reported by Genebank.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392.1

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