【摘要】：目的 利用重疊PCR合成風疹病毒E1全長基因,同時擴增E1主要抗原表位的基因序列,分別構(gòu)建它們的原核表達載體并表達蛋白,為研發(fā)高靈敏度風疹ELISA診斷試劑奠定基礎(chǔ)。 方法 對風疹E1基因進行生物信息學分析,根據(jù)大腸桿菌密碼子偏愛性對其密碼子進行優(yōu)化；設(shè)計多對寡核苷酸引物,以重疊PCR法分別合成該基因的3個片段,再用酶切連接法將各段拼接成全長為1 443 bp的RV E1,將E1基因的全長序列克隆導入原核表達載體pET32a獲得pET32-RV E1克隆并測序；選取E1全長基因中195 aa-305 aa抗原肽基因序列進行擴增,再以酶切連接的方法將合成的抗原肽序列克隆導入原核表達載體pET32a,獲得重組質(zhì)粒載體pET32-E1epitope測序鑒定。將重組原核表達載體pET32-RV El和pET32-E1epitope在大腸桿菌BL21(DE3)表達株中利用IPTG誘導蛋白表達并優(yōu)化蛋白表達量, SDS-PAGE和Western blot分析表達產(chǎn)物。 結(jié)果 1.分別進行了8輪、5輪和6輪的重疊PCR擴增,合成風疹E1全長基因3個片段；以酶切連接法將3個片段拼接成全長E1基因并克隆入pET32a,構(gòu)建載體pET32-RV E1,PCR、酶切和測序鑒定結(jié)果表明,合成的E1基因大小、序列與預期相符；構(gòu)建獲得重組質(zhì)粒pET32-RV E1。在37℃下用終濃度為1 mmol/L的IPTG誘導蛋白表達,Western blot檢測到預期的蛋白條帶； 2.以PCR擴增成功獲得風疹E1抗原肽基因并構(gòu)建重組質(zhì)粒pET32-El epitope,測序結(jié)果正確；在37℃下以終濃度為1 mmol/L IPTG誘導抗原肽表達,SDS-PAGE和Western blot檢測到預期的蛋白條帶；且以IPTG終濃度為0.1 mmol/L誘導6小時后表達量達到最高。 結(jié)論 成功合成了密碼子優(yōu)化的風疹病毒E1基因并構(gòu)建其重組質(zhì)粒pET32-RV E1,表達完整E1蛋白；同時成功擴增風疹病毒E1抗原肽段基因,構(gòu)建其原核表達載體pET32-E1epitope并表達該抗原肽。
[Abstract]:Objective to synthesize the full-length gene of rubella virus E1 by overlapping PCR and amplify the gene sequence of the major epitopes of E1, and construct their prokaryotic expression vectors and express proteins respectively. It lays a foundation for the development of high sensitivity ELISA diagnostic reagent for rubella. Methods the rubella E1 gene was analyzed by bioinformatics, its codon was optimized according to the codon preference of Escherichia coli, and three fragments of the gene were synthesized by overlapping PCR, and multiple pairs of oligonucleotide primers were designed. The whole sequence of E1 gene was cloned into prokaryotic expression vector pET32a to obtain pET32-RV E1 clone and sequenced, and 195 aa-305 aa antigenic peptide gene sequence of E1 full-length gene was amplified. The recombinant plasmid pET32-E1epitope was sequenced and cloned into prokaryotic expression vector pET32a, by restriction endonuclease ligation. The recombinant prokaryotic expression vectors pET32-RV El and pET32-E1epitope were induced by IPTG in E. coli BL21 (DE3) expression strain and the expression amount was optimized. The expression products were analyzed by SDS-PAGE and Western blot. Result 1. Three fragments of rubella E1 full-length gene were synthesized by overlapping PCR amplification of 8 rounds and 5 rounds and 6 rounds, respectively. The three fragments were spliced into full-length E1 gene by restriction endonuclease ligation and cloned into pET32a, to construct the vector pET32-RV E1 PCR. The results of restriction endonuclease digestion and sequencing showed that, The size of the synthesized E1 gene was consistent with the expected sequence, and the recombinant plasmid pET32-RV E1 was constructed. At 37 鈩，

本文編號：2249721