[1]卢春霞,刘长彬,石国庆,等.基于密码子优化策略的牛妊娠相关糖蛋白9(bPAG9)的原核表达及纯化[J].江苏农业学报,2020,(01):122-129.[doi:doi:10.3969/j.issn.1000-4440.2020.01.017]
 LU Chun-xia,LIU Chang-bin,SHI Guo-qing,et al.Prokaryotic expression and purification of recombinant bovine pregnancy associated glycoprotein-9 (bPAG9) based on codon optimization[J].,2020,(01):122-129.[doi:doi:10.3969/j.issn.1000-4440.2020.01.017]
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基于密码子优化策略的牛妊娠相关糖蛋白9(bPAG9)的原核表达及纯化()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年01期
页码:
122-129
栏目:
畜牧兽医·水产养殖
出版日期:
2020-02-29

文章信息/Info

Title:
Prokaryotic expression and purification of recombinant bovine pregnancy associated glycoprotein-9 (bPAG9) based on codon optimization
作者:
卢春霞1刘长彬23石国庆3杨华3
(1.长江师范学院现代农业与生物工程学院,重庆408100;2.石河子大学动物科技学院,新疆石河子832003;3.新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子83200)
Author(s):
LU Chun-xia1LIU Chang-bin23SHI Guo-qing3YANG Hua3
(1.School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China;2.College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;3.Key Laboratories of Sheep Breeding and Reproduce, Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China)
关键词:
牛妊娠相关糖蛋白密码子重组蛋白原核表达
Keywords:
bovine pregnancy associated glycoproteincodonrecombinant proteinprokaryotic expression
分类号:
S823.9+13+.5
DOI:
doi:10.3969/j.issn.1000-4440.2020.01.017
文献标志码:
A
摘要:
为了构建pET30a-bPAG9重组表达载体,并在BL21(DE3) 感受态细胞中转染高效表达牛妊娠相关糖蛋白9(bPAG9),通过生物信息学技术对bPAG9基因进行密码子优化,人工合成bPAG9全基因序列,经双酶切法将bPAG9基因插入到表达载体pET30a中。将构建的重组质粒pET30a-bPAG9转染至BL21(DE3) 感受态细胞中进行原核表达,经过超声波裂解和亲和层析方法获得重组蛋白bPAG9。用SDS-PAGE 和 Western Blot方法检测重组蛋白bPAG9的表达效果。结果显示,PCR扩增得到1 128 bp的优化bPAG9基因片段,构建的重组质粒pET30a-bPAG9经双酶切获得约5 244 bp和1 125 bp的2条片段,与预期值相符;对重组载体测序,测序结果与优化后的基因碱基序列完全一致,编码的氨基酸序列未发生突变;SDS-PAGE 和 Western Blot 鉴定结果显示,获得相对分子质量约为4.0×104的bPAG9重组蛋白,通过亲和层析纯化后,重组蛋白bPAG9纯度到达90%以上。
Abstract:
To construct the recombinant expression vector pET30a-bPAG9 and express bovine pregnancy associated glycorprotein-9(bPAG9) in BL21(DE3) cells, the codons of original bPAG9 gene were redesigned and optimized by bioinformatics techniques. Furthermore, the codon-optimized gene bPAG9 was synthesized by PCR and connected with pET30a vector by double digestion. The pET30a-bPAG9 expression vector was constructed and transfected into BL21(DE3) cells. The recombinant protein bPAG9 was obtained by ultrasonic lysis and affinity chromatography. SDS-PAGE and Western blotting were performed to detect the expression of the recombinant protein bPAG9. The results showed that the optimized bPAG9 gene fragment of 1 128 bp was obtained by PCR. After amplification and enzymatic digestion, two fragments of 5 244 bp and 1 125 bp were obtained from pET30a-bPAG9 expression vector. These results were consistent with expectations. The sequencing results of recombinant vector showed that the base sequence of bPAG9 gene in expression vector was consistent with the optimized gene, and the amino acid was not mutated. The results Westernblotting and SDS-PAGE indicated that the recombinant protein bPAG9 with the relative molecular weight of about 4.0×104 was successfully expressed in BL21(DE3) cells. The purity of recombinant protein bPAG9 was over 90% after purification by affinity chromatography.

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备注/Memo

备注/Memo:
收稿日期:2019-05-09基金项目:国家自然科学基金项目(31860647)作者简介:卢春霞(1978-),女,河南虞城人,博士,副研究员,主要从事农产品质量安全研究。 (E-mail)shzlcx2002@163.com通讯作者:刘长彬, (E-mail)xlchangbin@163.com
更新日期/Last Update: 2020-03-13