Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (10): 143-151.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1401
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HU Xiao(), WANG Bao-bao, DOU Shao-hua, JIANG Nan, FU Chang-zhen, JIN Hang, GAO Feng-shan()
Received:
2020-11-17
Online:
2021-10-26
Published:
2021-11-12
Contact:
GAO Feng-shan
E-mail:eeeeyouxiang@163.com;gfsh0626@126.com
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.
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 |
pSLA-2-YT-R | GTTGCGGCCGCTCACACTCTAGGATCCTTGGTAAGGGACAC | Not I |
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 |
pSLA-2-YT-R | GTTGCGGCCGCTCACACTCTAGGATCCTTGGTAAGGGACAC | Not I |
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. 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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