Construction of a Eukaryotic Expression Vector of SLA-2 Gene from Yantai Black Pigs and Its Expression

doi:10.13560/j.cnki.biotech.bull.1985.2020-1401

Abstract

Abstract:

In order to construct the recombinant eukaryotic expression vector SLA-2-YT/pCDH from a Yantai black pig’s SLA-2 gene（SLA-2-YT）and to express it in eukaryotic cells,a pair of primers were designed according to SLA-2-YT coding region gene sequence,and the SLA-2-YT coding region gene fragment was amplified by PCR using SLA-2-YT/pMD18-T whole-gene cloning expression vector. The restriction enzyme Xba I and Not I were added to the 5'end of the forward and lower primers,respectively. The target gene was firstly cloned into pMD 19-T Simple Vector TA and was ligated to the pCDH-CMV-MCS-EF1-Puro（pCDH）eukaryotic expression vector. The recombinant plasmids were transformed into Escherichia coli Stbl 3 competent cells. The recombinant plasmids were extracted from the expanded culturing monoclonal strain and the sequences were verified by double enzyme digestion and sequencing. A large quantity of endotoxin-free plasmids was extracted from the recombinant eukaryotic expression vector with correct inserting sequence,and then they were transfected into sT2 cells through lentivirus packaging and infection. The expression of SLA-2-YT gene in sT2 cells was detected by Western Blotting. The results showed that the recombinant eukaryotic expression vector SLA-2-YT/pCDH was successfully constructed. After packaging by lentivirus,the recombinant plasmids infected sT2 cells followed by puromycin screening,and then a positive cell clone was successfully obtained. Western Blotting detection showed that SLA-2-YT/pCDH was predominantly expressed in sT2 cells with the molecular weight of 45 kD,which was consistent with the theoretical designing value. In this experiment,the SLA-2-YT coding region gene was successfully constructed into the eukaryotic expression vector,and predominantly expressed in sT2 cells,which will supply good materials for studying CTL epitoes presented by SLA-2-YT in future.

Key words: Yantai black pig, SLA-2 gene, eukaryotic expression vector, lentivirus packaging, transfection

HU Xiao, WANG Bao-bao, DOU Shao-hua, JIANG Nan, FU Chang-zhen, JIN Hang, GAO Feng-shan. Construction of a Eukaryotic Expression Vector of SLA-2 Gene from Yantai Black Pigs and Its Expression[J]. Biotechnology Bulletin, 2021, 37(10): 143-151.

Figures/Tables 10

Table 1 Information of the primers used in PCR

基因 Gene	登录号 GenBank accession No.	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	限制性内切酶 Cleavage sites of restriction enzyme
SLA-2-YT	AB672508.1T	pSLA-2-YT-F	GCTCTAGAATGCGGGTCAGGGGCCCTCAAGCCATCCTC	Xba I
SLA-2-YT	AB672508.1T	pSLA-2-YT-R	GTTGCGGCCGCTCACACTCTAGGATCCTTGGTAAGGGACAC	Not I

Fig. 1 Schematic diagram of the recombinant expression plasmid SLA-2-YT/pCDH

Fig. 2 PCR amplification and tailing of SLA-2-YT gene CDS region in Yantai black pig A is the PCR amplified product of SLA-2-YT gene CDS region of Yantai black pig; M: DNA marker 2000; 1: The PCR productsed of the CDS region of SLA-2-YT gene. B is the detection map of PCR amplificated products plus poly A tail; M: DNA marker 2000, 1: Recovery of PCR products after tailing

Fig. 3 Identification of recombinant plasmid SLA-2-YT/ pMD19-T Simple by double digestion M: DNA marker 2000, 1-7: double digestion products of recombinant plasmid SLA-2-YT/pMD19-T

Fig. 4 Insertion sequences in SLA-2-YT/pMD 19-T Simple recombinant vector analyzed by Vector NTI 11.5

Fig. 5 pCDH-CMV-MCS-EF1-Puro vector recovery M:DNA marker 10000, and 1: the recovered product of pCDH-CMV-MCSEF1- Puro vector digested by Xba I and Not I

Fig. 6 Identification of recombinant plasmid SLA-2-YT/pCDH by double digestion M:DNA marker 10000, and 1-3: double digestion products of recombinant plasmid SLA-2-YT/pCDH

Fig. 7 Lethal concentration curve of puromycin on sT2 cells The x-axis is the concentration of puromycin, and the y-axis is the number of living cells in the plate

Fig. 8 Observation of cell state after lentivirus infection under light microscope A is PK15 cell, B is sT2 cell, C is sT2 cell transfected with pCDH-CMVMCS-EF1-Puro, D is sT2 cell transfected with SLA-2-YT/pCDH

Fig. 9 Western Blotting to detect the expression of SLA-2-YT/pCDH in sT2 cells A is the result of Western blotting. The expression of SLA-2-YT/pCDH protein (45 kD) is measured with GAPDH (36 kD) as internal parameter (**P<0.01)

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