β2m基因在猪肾细胞系PK-15中的表达研究

doi:10.16431/j.cnki.1671-7236.2020.02.007

摘要/Abstract

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EcoR Ⅰ和Hind Ⅲ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。

关键词: 猪肾细胞; PK-15; β2m; 克隆; 表达

Abstract: To study the expression of β2m gene in swine kidney PK-15 cell line,total RNA was extracted and β2m was amplified by RT-PCR.The recovered gene product was cloned into pMD18-T vector to get some recombinant plasmids.After being screened by EcoR Ⅰ and Hind Ⅲ double enzyme digestion,the positive clones were sequenced in a biological company.The sequence of the β2m 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 the β2m from PK-15 with other animal β2m was constructed by NJ method of Mega 5.0.The secondary structure and tertiary structure of β2m 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 of β2m by Western blotting.The results showed that β2m gene was amplified successfully by RT-PCR.The fragment was about 360 bp.After sequencing,it showed that the real length of β2m 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 of β2m gene in PK-15 cells.The phylogenetic tree showed that PK-15-β2m was the closest to bovine β2m,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 equine β2m was constituted by 98 amino acids,while 99 amino acids in other animal β2m.The secondary structure prediction showed that the mature peptide of PK-15-β2m was mainly composed of β-sheet,β-turn and γ-turn,without α-helix.The tertiary structure prediction showed that the mature peptide of PK-15-β2m was constituted by 7 β-sheet.The protein samples isolated from PK-15 cells were detected by Western blotting and it was proved that β2m was successfully expressed in the PK-15 cells.This study confirmed the stable expression of β2m in nucleic acid and protein level in PK-15 cell line,which laid a foundation for the further study of the antigen-presenting function of PK-15 cells.

Key words: swine kidney cells; PK-15; β2m; cloning; expression

中图分类号:

S852.4

张宗辉, 王心怡, 曹海虹, 胡晓, 王宝宝, 高凤山. β2m基因在猪肾细胞系PK-15中的表达研究[J]. 中国畜牧兽医, 2020, 47(2): 372-380.

ZHANG Zonghui, WANG Xinyi, CAO Haihong, HU Xiao, WANG Baobao, GAO Fengshan. A Study on the Expression of β2m Gene in Swine Kidney Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 372-380.

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