β2m基因在猪肾细胞系PK-15中的表达研究  

Study on the Expression of β2m Gene in Swine Kidney Cells

作　　者：张宗辉 王心怡 曹海虹 胡晓 王宝宝 高凤山[1] ZHANG Zonghui;WANG Xinyi;CAO Haihong;HU Xiao;WANG Baobao;GAO Fengshan(College of Life Science and Technology,Dalian University,Dalian 116622,China;Zhuolu Agricultural and Rural Burea in Hebei Province,Zhuolu 075600,China)

机构地区：[1]大连大学生命科学与技术学院,大连116622 [2]河北省涿鹿县农业农村局,涿鹿075600

出　　处：《中国畜牧兽医》2020年第2期372-380,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目:CRISPR/Cas9技术构建sT2细胞系筛选猪源病毒SLA-I限制性CTL表位的研究(31672525);辽宁省教育厅重点实验室项目(LZ2015003);大连大学创新创业训练计划项目(2018158)

摘　　要：为研究猪肾细胞系PK-15中Beta 2微球蛋白(Beta 2 microglobulin,β2m)基因的表达情况,提取细胞总RNA,经RT-PCR扩增β2m。回收后的基因克隆至pMD18-T载体,获得重组质粒,经Eco RⅠ和Hin dⅢ双酶切筛选后,阳性克隆送生物公司测序。序列经GENETYX version 9.0编辑分析,通过DNAMAN version 5.2.2与其他猪β2m基因进行序列比对分析,利用Mega 5.0的NJ法进一步分析其与其他物种β2m的分子进化关系,并在此基础上利用SWISS-MODEL和PDBsum预测该基因编码的成熟肽二级结构和三级结构。提取PK-15总蛋白,Western blotting检测和分析细胞中β2m的表达。结果显示,经RT-PCR扩增,成功获得β2m条带,大小约360 bp。经测序发现PK-15-β2m基因为364 bp,共编码118个氨基酸,其中成熟肽为98个氨基酸,N端信号肽含有20个氨基酸。序列分析证实PK-15细胞中β2m基因未发生突变;分子进化分析显示,PK-15-β2m与牛的亲缘关系最近,其次为羊、马等。多重序列比对发现,PK-15-β2m与牛、羊、马β2m成熟肽均为98个氨基酸,而其他物种β2m成熟肽为99个氨基酸;二级结构预测显示,PK-15-β2m成熟肽主要以β-折叠、β-转角和γ-转角为主,不含有α-螺旋。三级结构预测显示,PK-15-β2m成熟肽主要由7条β-折叠条带构成。Western blotting分析显示β2m在细胞中成功表达。本研究证实了猪肾细胞系PK-15中β2m在核酸和蛋白质水平均稳定表达,为下一步研究PK-15细胞的抗原递呈功能奠定了基础。To study the expression ofβ2m gene in swine kidney PK-15 cell line,total RNA was extracted andβ2m was amplified by RT-PCR.The recovered gene product was cloned into pMD18-T vector to get some recombinant plasmids.After being screened by Eco RⅠand Hin dⅢdouble enzyme digestion,the positive clones were sequenced in a biological company.The sequence of theβ2m from PK-15 was edited and analyzed by GENETYX version 9.0,then,it was compared with other swine by DNAMAN version 5.2.2.The phylogenetic tree of theβ2m from PK-15 with other animalβ2m was constructed by NJ method of Mega 5.0.The secondary structure and tertiary structure ofβ2m from PK-15 were also predicted and analyzed by SWISS-MODEL and PDBsum.Proteins extracted from PK-15 cells were used to detect the expression ofβ2m by Western blotting.The results showed thatβ2m gene was amplified successfully by RT-PCR.The fragment was about 360 bp.After sequencing,it showed that the real length ofβ2m gene was 364 bp coded 118 amino acids including a mature peptide with 98 amino acids on the carboxyl terminal and a signal peptide with 20 amino acids on the amino terminal.Sequences analyzing confirmed that there was no mutation ofβ2m gene in PK-15 cells.The phylogenetic tree showed that PK-15-β2m was the closest to bovineβ2m,followed by sheep,horses,etc.The results of multiple sequence comparison showed that the mature peptide of PK-15-β2m along with other bovine,ovine and equineβ2m was constituted by 98 amino acids,while 99 amino acids in other animalβ2m.The secondary structure prediction showed that the mature peptide of PK-15-β2m was mainly composed ofβ-sheet,β-turn andγ-turn,withoutα-helix.The tertiary structure prediction showed that the mature peptide of PK-15-β2m was constituted by 7β-sheet.The protein samples isolated from PK-15 cells were detected by Western blotting and it was proved thatβ2m was successfully expressed in the PK-15 cells.This study confirmed the stable expression ofβ2m in nucleic acid and protein level in PK-15 cell lin

关 键 词：猪肾细胞 PK-15 Β2M 克隆 表达 

分 类 号：S852.4[农业科学—基础兽医学]