牛偏爱密码子优化后的Beta蛋白多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2015.10.008

摘要/Abstract

摘要：

本研究旨在优化λ噬菌体bet基因，使其能够在牛细胞中高效表达，通过密码子优化及全基因合成方法制备牛bet基因，并进行原核表达和多克隆抗体制备及抗体效价评估。从GenBank中获得λ噬菌体bet基因的序列，登录号为AP005153，用牛偏爱密码子对其进行优化并将其合成。根据优化后的基因序列设计引物，用PCR扩增优化后的bet基因，随后将其克隆到pET28原核表达载体，并转化入大肠杆菌BL21中进行诱导表达。表达的蛋白产物利用亲和层析的方法进行纯化，用His tag 的抗体进行Western blot 鉴定。用纯化后的蛋白免疫CD1小鼠，得到抗Beta蛋白的血清，通过ELISA测定该多克隆抗体的滴度。结果表明：通过双酶切和测序，成功挑选出正确的原核表达质粒pET-bet；SDS-PAGE，电泳结果显示融合蛋白表达成功；用纯化后的蛋白免疫试验鼠后得到多克隆抗体，经Western blot 检测，该多克隆抗体能与His-Beta融合蛋白特异性结合；经ELISA方法检测该多克隆抗体的滴度为1∶41 700。成功地制备了Beta蛋白的多克隆抗体，为深入探索bet基因的生物学功能和在牛及大型哺乳动物中的应用奠定了基础。

Abstract:

This study aimed to optimize bet gene of bacteriophage λ so that it can be efficiently expressed in cattle cells. The optimized bet gene was synthesized by genome-wide synthesis method and expressed in prokaryotic expressing vector，the polyclonal antibody was prepared and antibody titer evaluation was preformed.The sequence of bacteriophage λ bet gene was obtained from GenBank(GenBank accession No.:AP005153 ) and optimized with bovine preferred codons. Then synthesized the optimized gene.According to the optimized gene sequence,a pair of primers with the restriction enzyme sites were designed.The bet gene was amplified by PCR and cloned into the pET28a prokaryotic expressing vector. Fusion protein expressed in E.coli BL21 by IPTG induction,then we purified the protein products by affinity chromatography methods and identified by Western blot with His tag antibody. CD1 mice were immunized with the purified fusion protein. Serum with anti-Beta protein polyclonal antibody was obtained, and then the antibody titer was detected by ELISA assay. As a result,confirmed by restriction enzyme digestion and sequencing, pET-bet prokaryotic expressing vector was successfully constructed; The fusion protein was high efficiently expressed, which was confirmed by SDS-PAGE; CD1 mice were immunized with the purified protein, the polyclonal anti-Beta antibody with 1∶41 700 titer was prepared and it could bind His-Beta fusion protein specifically, which was confirmed by ELISA assay and Western blot assay. The successful preparation of anti-Beta protein polyclonal antibody will lay a solid foundation for further studies of the biological functions and applications in bovine and other mammals with bet gene.

中图分类号:

S823.2

齐昱，邢燕平，周欢敏，房君，钱呈，王海涛. 牛偏爱密码子优化后的Beta蛋白多克隆抗体制备[J]. 畜牧兽医学报, 2015, 46(10): 1759-1765.

QI Yu，XING Yan-ping，ZHOU Huan-min，FANG Jun，QIAN Cheng，WANG Hai-tao. Preparation of Polyclonal Antibody against Beta Protein Optimized with Bovine Preferred Codons[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(10): 1759-1765.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2015.10.008

https://www.xmsyxb.com/CN/Y2015/V46/I10/1759

参考文献

［1］CARTER D M，RADDING C M.The role of exonuclease and β protein of phage λ in genetic recombination II Substrate specificity and the mode of action of lambda exonuclease［J］. J Biol Chem，1971，246( 8)：2502-2512.
［2］KOVALL R，MATTHEWS B W.Toroidal structure of lambda-exonuclease［J］.Science，1997，277(5333)：1824-1827.
［3］LIU Q，XUE Q.Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species［J］.J Genet，2005，84(1)：55-62.
［4］EYRE-WALKER A C.An analysis of codon usage in mammals：selection or mutation bias［J］J Mol Evol，1991，33(5)：442-449.
［5］ROCHA E P.Codon usage bias from tRNA’s point of view：redundancy，specialization，and efficient decoding for translation optimization［J］.Genome Res，2004，14(11)：2279-2286.
［6］XIA X.Maximizing transcription efficiency causes codon usage bias［J］.Genetics，1996，144(3)：1309-1320.
［7］KARU A E，SAKAKI Y，ECHOLS H，et al.The gamma protein specified by bacteriophage λ.Structure and inhibitory activity for the RecBC enzyme of Escherichia coli［J］.J Biol Chem，1975，250(18)：7377-7387.
［8］MUNIYAPPA K，RADDING C M.The homologous recombination system of phage λ.Pairing activities of β protein［J］.J Biol Chem，1986，261(16)：7472-7478.
［9］POTEETE A R.What makes the bacteriophage λ Red system useful for genetic engineering：Molecular mechanism and biological function［J］.FEMS Microbiol Lett， 2001，201(1)：9-14.
［10］MURPHY K C.Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli［J］. J Bacteriol，1998，180(8)：2063-2071.
［11］ELLIS H M，YU D，DITIZIO T，et al.High efficiency mutagenesis，repair，and engineering of chromosomal DNA using single stranded oligonucleotides［J］.Proc Natl Acad Sci USA，2001，98(12)：6742-6746.
［12］ZHANG Y，MUYRERS J P，TESDA G，et al.DNA cloning by homologous recombination in Escherichia coli［J］.Nat Biotechnol，2000，18(12)：1314-1317.
［13］万小颖，秦立志，施丽娟，等.兔白细胞介素-10基因的克隆、原核表达及其多克隆抗体的制备［J］.畜牧兽医学报，2013，44(5)：788-795.
WAN X Y，QIN L Z，SHI L Z，et al.Cloning，prokaryotic expression and polyclonal antibody preparation of rabbit IL-10 gene［J］. Acta Veterinaria et Zootechnica Sinica，2013，44(5)：788-795.(in Chinese)
［14］陈江涛，王光祥，吉艳红，等.猪膜联蛋白A2多克隆抗体的制备、亲和纯化与鉴定［J］.畜牧兽医学报，2010，41(11)：1502-1509.
CHEN J T，WANG G X，JI Y H，et al.Preparation affinity purification and identification of polyclonal antibody against pig Annexin A2［J］.Acta Veterinaria et Zootechnica Sinica，2010，41(11)：1502-1509.(in Chinese)

[1]	苗舒, 安济山, 王祚, 肖定福, 兰欣怡, 刘磊, 沈维军, 万发春. 亮氨酸通过PI3K-AKT信号通路促进牛成肌细胞的增殖[J]. 畜牧兽医学报, 2024, 55(1): 142-152.
[2]	牛一凡, 杨柏高, 张培培, 张航, 冯肖艺, 曹建华, 余洲, 郝海生, 杜卫华, 邹惠影, 朱化彬, 马友记, 赵学明. 牛胚胎基因组选择研究进展[J]. 畜牧兽医学报, 2023, 54(11): 4449-4457.
[3]	王文翔, 杜丽丽, 胡俊伟, 张琰翔, 马铭昊, 段蕊, 钱聪, 王欣玥, 李三禄, 张长庆, 张路培, 高雪, 徐凌洋, 李俊雅, 高会江. 基于转录组分析挖掘平凉红牛背最长肌肉质性状候选基因[J]. 畜牧兽医学报, 2023, 54(11): 4589-4604.
[4]	马浩然, 张路培, 金生云, 宝金山, 李红艳, 高会江, 徐凌洋, 王泽昭, 李俊雅. 利用高密度SNP芯片评估中国地方肉牛品种基因组亲缘关系[J]. 畜牧兽医学报, 2023, 54(10): 4174-4185.
[5]	张俊星, 张海亮, 韩丽云, 马燕芬, 温万, 周佳敏, 田佳, 路婷婷, 马云, 王雅春. 宁夏地区荷斯坦泌乳牛健康性状影响因素分析[J]. 畜牧兽医学报, 2023, 54(6): 2389-2401.
[6]	杨闯, 吴龙飞, 柳广斌, 李耀坤, 刘德武, 孙宝丽. 雷琼牛与陆丰牛背最长肌lncRNA表达特点及其相关ceRNA网络分析[J]. 畜牧兽医学报, 2023, 54(5): 1951-1963.
[7]	呙明鹏, 孟源, 王宏浩, 王元, 车雷杰, 申莉, 王曦. 晋南牛不同生长阶段体重和体尺性状遗传参数估计[J]. 畜牧兽医学报, 2023, 54(4): 1452-1464.
[8]	余诗强, 李留学, 赵小博, 赵慧颖, 屠焰, 赵玉超, 蒋林树. 不同泌乳阶段和体细胞水平的中国荷斯坦奶牛泌乳性能差异和相关性研究[J]. 畜牧兽医学报, 2023, 54(3): 1003-1014.
[9]	杨光, 徐景, 李新, 胡德宝, 郭益文, 丁向彬, 郭宏, 张林林. 干扰lncbMD对牛骨骼肌卫星细胞增殖分化的影响[J]. 畜牧兽医学报, 2023, 54(3): 1015-1025.
[10]	马志杰, 王世康, 张卫忠, 郭卫兴, 赵海明, 雷初朝. 柴达木牛和蒙古牛Y染色体基因组遗传多样性与父系起源研究[J]. 畜牧兽医学报, 2023, 54(3): 1026-1033.
[11]	牛锐利, 宋哈楠, 吴月, 王育南, 高也凡, 关伟军, 宗宪春. 西门塔尔牛表皮干细胞的分离培养与生物学特性研究[J]. 畜牧兽医学报, 2023, 54(1): 168-177.
[12]	李宏伟, 徐凌洋, 王泽昭, 蔡文涛, 朱波, 陈燕, 高雪, 张路培, 高会江, 李俊雅. 基于单倍型肉牛屠宰性状全基因组关联分析研究[J]. 畜牧兽医学报, 2022, 53(12): 4232-4243.
[13]	常瑶, 苏国生, 李艳华, 李想, 麻柱, 王雅春. 基于系谱和基因组信息估计荷斯坦青年母牛体重性状遗传参数[J]. 畜牧兽医学报, 2022, 53(11): 3759-3768.
[14]	许静漪, 徐昊祺, 胡丽蓉, 张帆, 高清, 罗汉鹏, 张海亮, 师睿, 李想, 刘林, 郭刚, 王雅春. MET基因多态性与中国荷斯坦牛繁殖和泌乳性状的关联分析[J]. 畜牧兽医学报, 2022, 53(11): 3769-3785.
[15]	王川川, 母童, 冯小芳, 禹保军, 张娟, 顾亚玲. 基于转录组测序挖掘奶牛乳脂代谢关键候选基因[J]. 畜牧兽医学报, 2022, 53(10): 3421-3433.