烟台黑猪SLA-I 重链基因末端生物素修饰及表达

生物技术通报 ›› 2014, Vol. 0 ›› Issue (1): 191-195.

烟台黑猪SLA-I 重链基因末端生物素修饰及表达

刘筏,杨金刚,翟晓鑫,董宋鹏,高凤山

大连大学生命科学与技术学院，大连 116622

收稿日期:2013-09-11 出版日期:2014-01-23 发布日期:2014-01-23
作者简介:刘筏，女，研究方向: 动物分子免疫学；E-mail: 729652171@qq.com
基金资助:
国家自然科学基金项目(31172304)，辽宁省大学生创新项目(201311258009)，大连大学本科生创新项目(2012029)

Biotinylated the Carboxyl Terminal of Heavy Chain of SLA-I from Yantai Black Pig and Its Expression

Liu Fa, Yang Jingang, Zhai Xiaoxin, Dong Songpeng, Gao Fengshan

College of Life Science and Technology，Dalian University，Dalian 116622

Received:2013-09-11 Published:2014-01-23 Online:2014-01-23

摘要/Abstract

摘要： 为构建烟台黑猪SLA-2 重链基因偶合生物素化酶BirA 底物肽(BirA substrate peptide，BSP)并研究其在pET-28a#br#(+)中的表达，设计烟台黑猪SLA-2-BSP 复合基因引物，PCR 扩增烟台黑猪SLA-2-YTH-BSP 复合基因，并克隆至pMD 19-T Simple#br#Vectorp，经酶切鉴定后将SLA-2-YTH-BSP 复合基因与表达系统pET-28a(+)链接，并转化到BL21(Rosseta)菌进行诱导表达，#br#SDS-PAGE 检测目的蛋白。表达蛋白经过Ni-NTA 亲和纯化，并经SDS-PAGE 检测蛋白纯化效果。PCR 结果显示SLA-2-BSP 大小为#br#900 bp，并成功克隆到pMD 19-T Simple Vector，酶切鉴定大小为876 bp，该基因链接到 pET-28a(+)并转化到BL21(Rosseta)菌，#br#经诱导表达和SDS-PAGE 检测目的蛋白的大小为32.4 kD。目的蛋白以包涵体形式存在，纯化后蛋白纯度达到90% 以上。成功构建#br#烟台黑猪SLA-I 重链偶合生物素化酶BirA 底物肽(BSP)的pET-28a(+)的重组表达系，为下一步的SLA-I 类分子四聚体研究鉴#br#定基础。

关键词: 烟台黑猪 SLA-2, 重链基因, 生物素化酶BirA, 底物肽, 纯化

Abstract: To construct the heavy chain of SLA-2 conjugated with the BirA substrate peptide(BSP)and express the recombinant genes in pET-28a(+)for Yantai Black Pig(YTH), a pair of primers to express the recombinant SLA-2-YTH-BSP was designed and the recombinant SLA-2-YTH-BSP was amplified by PCR followed by sub-cloning the gene into pMD19-T Simple Vector. After identification by cleavage with Nde I and Xho I, the SLA-2-YTH-BSP was ligated to pET-28a(+)and the recombinant plasmids was transformed into BL21 (Rosseta)to be induced to express followed by analysis of the expressing products by SDS-PAGE. The expressed interest of protein was purified by Ni-NTA column and detected by SDS-PAGE. The PCR results showed that the length of nucleotides of SLA-2-YTH-BSP was about 900 bp which was consistent with the calculated value. Then, the SLA-2-YTH-BSP with 876 bp was successfully inserted into the pMD-19-T Simple Vector identified by cleavage with Nde I and Xho I, and then the genes were inserted into pET-28a(+)and transformed into Escherichia coli BL21(Rosseta). After induction, SLA-2-YTH-BSP was successfully expressed with the interest of protein about 32.4 kD. After purification,the purity of the interest of the inclusion protein reached to 90%. It was concluded that the recombinant expressing system containing SLA-2 conjugated with BSP in pET-28a(+)was successfully constructed and the research would pave the base to construct tetramer in SLA class I.

Key words: Yantai black pig SLA-2, Heavy chain gene, BirA substrate peptide, Purification

刘筏,杨金刚,翟晓鑫,董宋鹏,高凤山. 烟台黑猪SLA-I 重链基因末端生物素修饰及表达[J]. 生物技术通报, 2014, 0(1): 191-195.

Liu Fa, Yang Jingang, Zhai Xiaoxin, Dong Songpeng, Gao Fengshan. Biotinylated the Carboxyl Terminal of Heavy Chain of SLA-I from Yantai Black Pig and Its Expression[J]. Biotechnology Bulletin, 2014, 0(1): 191-195.

参考文献

[1] Lemke CD, Graham JB, Lubaroff DM, et al. Development of an MHC class I L(d)-restricted PSA peptide-loaded tetramer for detection of PSA-specific CD8+ T cells in the mouse[J]. Prostate Cancer Prostatic Dis, 2011, 14(2): 118-121.
[2] Jiang X, Lu X, Liu R, et al. HLA Tetramer based artificial antigenpresenting cells efficiently stimulate CTLs specific for malignant glioma[J]. Clin Cancer Res, 2007, 13(24): 7329-7334.
[3] Savage P, Millrain M, Dimakou S, et al. Expansion of CD8+ cytotoxic T cells in vitro and in vivo using MHC class I tetramers[J]. Tumour Biol, 2007, 28(2): 70-76.
[4] Liu G, Wang Q, Tong T, et al. Construction and functional test of a chicken MHC-I(BF2*15)/peptide tetramer[J]. Vet Immunol Immunopathol, 2008, 122(1-2): 1-7.
[5] 张香春, 冯磊, 张鑫, 等. 荷包猪SLA 重链基因偶合底物肽及表达研究[J]. 生物技术, 2012, 22(5): 17-20.
[6] 许刚璞, 许瑜伟. 烟台黑猪的调查报告[J]. 中国畜禽种业 2011(6): 61-62.
[7] 白婧, 李云岗, 张强, 等. 六品系猪SLA-2 多肽结合区分子结构差异研究[J]. 宁夏大学学报, 2012, 33(3): 274-278.
[8] 高凤山, 夏春, 张强, 等. 大肠杆菌表达的重组猪β2 微球蛋白二级结构的圆二色谱分析[J]. 微生物学报, 2009, 49(12): 1596-1600.
[9] 郑秋实, 段晨曦, 陶凤云. SMAP-29 抗菌肽的原核表达、纯化及活性检测[J]. 生物技术通报, 2013(3): 144-148.
[10] Yokouchi H, Chamoto K, Wakita D, et al. Tetramer-blocking assay for defining antigen-specific cytotoxic T lymphocytes using peptide-MHC tetramer[J]. Cancer Sci, 2006, 97(2): 148 -154.
[11] Ouyang D, Wang X, He X, et al. Construction of soluble Mamub* 1703, a class I major histocompatibility complex of Chinese rhesus macaques, monomer and tetramer loaded with a simian immunodeficiency virus peptide[J]. Cell Mol Immunol, 2009, 6 (2): 117-122.

[1]	李文硕, 王林松, 杜桂彩, 郭群群, 张廷婷, 杨宏, 李荣贵. 一种松材线虫醛脱氢酶的基因克隆及其生化性质[J]. 生物技术通报, 2022, 38(9): 207-214.
[2]	覃雪晶, 王雨涵, 曹一博, 张凌云. 青杄PwHAP5基因原核表达及多克隆抗体制备[J]. 生物技术通报, 2022, 38(8): 142-149.
[3]	易芳, 来鹏程, 郑希鳌, 胡帅, 高燕丽. Kod DNA聚合酶的制备及纯化研究[J]. 生物技术通报, 2022, 38(5): 183-190.
[4]	王小琴, 黄银萍, 王蔚倩, 吴萍, 全舒. 含非天然氨基酸定点突变的MLL3_SET蛋白表达与纯化[J]. 生物技术通报, 2022, 38(3): 194-202.
[5]	张丰文, 周丽亚, 董超, 史延茂. 纳豆中抗氧化肽的分离纯化与活性研究[J]. 生物技术通报, 2022, 38(2): 158-165.
[6]	陈铎, 刘永哲. 新型冠状病毒核蛋白N端结构域的表达纯化以及晶体优化[J]. 生物技术通报, 2022, 38(12): 149-155.
[7]	曹汝菲, 李泽轩, 许欢, 张莎, 张敏敏, 戴枫, 段晓雷. 脆弱拟杆菌Pif1解旋酶的表达纯化与晶体生长[J]. 生物技术通报, 2021, 37(9): 180-190.
[8]	田嘉慧, 封佳丽, 卢俊桦, 毛林静, 胡著然, 王莹, 楚杰. 一色齿毛菌漆酶LacT-1的分离纯化与性质研究[J]. 生物技术通报, 2021, 37(8): 186-194.
[9]	张西西, 张怡青, 李玉林, 韩笑, 王国强, 王晓军, 王旭东, 王云龙. 新型冠状病毒（SARS-CoV-2）N蛋白C端重组蛋白的原核表达、纯化及应用[J]. 生物技术通报, 2021, 37(5): 92-97.
[10]	王志新, 鲁雷震, 周景波, 封成玲, 贾紫伟, 宁亚维, 贾英民. 抗真菌肽研究进展[J]. 生物技术通报, 2021, 37(3): 206-218.
[11]	唐禄, 董丽平, 尹茉莉, 刘磊, 董媛, 王会岩. 成纤维细胞生长因子20单克隆抗体的制备及鉴定[J]. 生物技术通报, 2021, 37(10): 179-185.
[12]	孟利, 杜彩萍. 大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定[J]. 生物技术通报, 2020, 36(12): 98-103.
[13]	周阳, 王桃桃, 闫丹丹, 王莹莹, 施沁璇, 孙丽慧, 林锋. 类弹性蛋白作为功能纳米材料在生物工程领域研究及应用进展[J]. 生物技术通报, 2020, 36(11): 198-208.
[14]	段佳琪, 李琲琲, 任杰, 邱德文, 李广悦. 一种脱氧雪腐镰刀菌烯醇制备工艺的研究[J]. 生物技术通报, 2020, 36(10): 256-262.
[15]	吴怡, 马鸿飞, 曹永佳, 司静, 崔宝凯. 真菌漆酶的性质、生产、纯化及固定化研究进展[J]. 生物技术通报, 2019, 35(9): 1-10.