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huojinlong8610金虫 (小有名气)
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[求助]
润色英文
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为了获得猪白细胞介素6（IL6）和α干扰素（IFNα）双重活性的融合蛋白，研究其作为免疫佐剂的可行性，利用IL6和IFNα基因的编码区序列以及原核表达载体pET32a序列，设计了带有限制性酶切位点、Linker序列以及His标签的特异性引物扩增出了猪IL6和IFNα的成熟肽基因序列，并分别与克隆载体pMD18连接后转化E.coli DH5α，提取质粒酶切并连接构建重组质粒pMD18-IL6-IFNα，将该重组质粒和表达载体pET32a同时酶切并连接构建pET32-IL6-IFNα重组质粒，依次转化E.coli DH5α和E.coli Rosetta (DE3)感受态细胞，并经不同浓度的IPTG以及不同时间进行诱导表达。结果成功构建了IL6和IFNα的融合表达载体，SDS-PAGE检测pET32-IL6-IFNα融合蛋白在E.coli Rosetta (DE3)中得到了较高表达，目的蛋白的相对分子量为44.96 KDa，与理论预期值一致，超声波后SDS-PAGE检测发现蛋白主要以不溶性的包涵体形式存在。 To explore the feasibility of interleukin-6/interferon-α (IL6/IFNα) fusion protein as an immunoadjuvant, the coding sequences of IL6 and IFNα were cloned. Considering the characteristics of these coding sequences and the sequence of prokaryotic expression vector pET32a, the mature peptide sequences of IL6 and IFNα were amplified using the specific primers designed with restriction enzyme sites, linker sequences and His tag, and then transfected into E.coli DH5α after linked with pMD18, respectively. The plasmids were extracted and digested by restriction enzyme respectively, and linked to construct the recombinant plasmid of pMD18-IL6-IFNα. The plasmid was transformed into E.coli DH5α and Rosetta (DE3), and then expressed under different concentrations' IPTG and times. The results showed that the recombinant plasmid of pET32-IL6-IFNα was constructed successfully, SDS-PAGE results showed that recombinant fusion proteins were expressed highly in E.coli Rosetta (DE3), and the molecular weight of inclusion bodies is 44.96 KDa. pET32a-IL6-IFNα was detected after ultrasonic treatment by PAGE and found existed as inclusion body form. |
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yangruyiyantai
木虫 (著名写手)
- 翻译EPI: 8
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★ ★ ★ ★ ★
爱与雨下: 金币+1 2013-03-19 21:08:20
huojinlong8610: 金币+5, 翻译EPI+1, 没有进行润色，照搬了我原来的 2013-03-20 15:33:23
phu_grassman: 金币-1, 翻译EPI-1, 未进行润色，撤销EPI。 2013-04-04 09:20:15
爱与雨下: 金币+1 2013-03-19 21:08:20
huojinlong8610: 金币+5, 翻译EPI+1, 没有进行润色，照搬了我原来的 2013-03-20 15:33:23
phu_grassman: 金币-1, 翻译EPI-1, 未进行润色，撤销EPI。 2013-04-04 09:20:15
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To aquire the feasibility of interleukin-6/interferon-α (IL6/IFNα) fusion protein as an immunoadjuvant, the coding sequences of IL6 and IFNα were cloned. Considering the characteristics of these coding sequences and the sequence of prokaryotic expression vector pET32a, the mature peptide sequences of IL6 and IFNα were amplified using the specific primers designed with restriction enzyme sites, linker sequences and His tag, and then transfected into E.coli DH5α after linked with pMD18, respectively. The plasmids were extracted and digested by restriction enzyme respectively, and linked to construct the recombinant plasmid of pMD18-IL6-IFNα. The plasmid was transformed into E.coli DH5α and Rosetta (DE3), and then expressed under different concentrations' IPTG and times. The results showed that the recombinant plasmid of pET32-IL6-IFNα was constructed successfully, SDS-PAGE results showed that recombinant fusion proteins were expressed highly in E.coli Rosetta (DE3), and the relative molecular weight of inclusion bodies is 44.96 KDa. pET32a-IL6-IFNα was detected after ultrasonic treatment by PAGE and found existing as inclusion body form. |
2楼2013-03-19 17:27:45
cory0931
至尊木虫 (文坛精英)
巫山云
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【答案】应助回帖
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爱与雨下: 金币+1 2013-03-19 21:08:24
huojinlong8610: 金币+50, ★有帮助 2013-03-20 15:40:47
phu_grassman: 金币+1, 翻译EPI+1, thanks for your help/ 2013-04-04 09:21:21
爱与雨下: 金币+1 2013-03-19 21:08:24
huojinlong8610: 金币+50, ★有帮助 2013-03-20 15:40:47
phu_grassman: 金币+1, 翻译EPI+1, thanks for your help/ 2013-04-04 09:21:21
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小试一下，仅供参考； To obtain the fusion protein of swine interleukin-6 & interferon-α(IL6 & IFNα) and explore its feasibility of using as an immunoadjuvant, primers with restriction endonuclease sites, linker and his-tag was designed according to the sequences of Il6/IFNα and vector pET32a. Matured peptide sequences of swine IL6 & IFNαwere successfully cloned and amplified by transfecting into E. coli through pMD18, respectively. The pMD18 plasmids were extracted and enzymatically cut to build the recombinant plasmid pMD18-IL6-IFNα(rpMD18-IL6-IFNα), followed by transfection into competent E. coli DH5α and E. coli Rosetta(DE3), and expressed by different IPTG concentration induction in different time. The results showed that the fusion expression plasmids were successfully built and highly expressed in E. coli Rosetta(DE3) according to SDS-PAGE detection. Molecular weight of the interest protein is 44.96kDa, which is agree with predicted value, and is mainly existed as insoluble inclusion bodies by SDS-PAGE detection after ultrasound treatment. |
3楼2013-03-19 19:34:18
hookhans
铁杆木虫 (著名写手)
Farmer
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4楼2013-03-19 19:40:46
