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

Abstract

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

CLC Number:

S852.4

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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A Study on the Expression of β2m Gene in Swine Kidney Cells

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