烟台黑猪SLA-2基因真核表达载体的构建及表达

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

摘要/Abstract

摘要：

为构建烟台黑猪SLA-2基因（SLA-2-YT）的真核表达载体SLA-2-YT/pCDH并将其在真核细胞中表达,根据SLA-2-YT编码区基因序列设计1对引物,以SLA-2-YT/pMD18-T全基因克隆表达载体为模板进行PCR扩增获得SLA-2-YT编码区基因片段,在上下游引物的5'端分别添加限制性内切酶Xba I和Not I的酶切位点,将目的基因经pMD 19-T Simple Vector TA克隆后与pCDH-CMV-MCS-EF1-Puro真核表达载体连接,获得的重组质粒转化至大肠杆菌Stbl 3感受态细胞,对扩大培养的单克隆菌株抽提质粒,使用双酶切和测序验证插入序列。对真核表达载体构建正确的菌株抽提无内毒素质粒,通过慢病毒包装和感染将质粒转染至sT2细胞,通过Western Blotting检测sT2细胞中SLA-2-YT基因的表达情况。结果显示,成功构建了SLA-2-YT/pCDH重组真核表达载体。重组质粒进行慢病毒包装和感染sT2细胞,经嘌呤霉素筛选后,成功获得阳性细胞克隆。Western Blotting检测显示SLA-2-YT/pCDH在sT2细胞中得到了优势表达,其融合蛋白的分子量大小为45 kD,与理论设计值相符。该实验成功构建了SLA-2-YT编码区基因的真核表达载体,并证实了SLA-2-YT编码区基因能够在sT2细胞中优势表达,为下一步开展SLA-2-YT递呈CTL表位的研究提供了材料。

关键词: 烟台黑猪, SLA-2基因, 真核表达载体, 慢病毒包装, 转染

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

胡晓, 王宝宝, 窦少华, 姜南, 付常振, 金行, 高凤山. 烟台黑猪SLA-2基因真核表达载体的构建及表达[J]. 生物技术通报, 2021, 37(10): 143-151.

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.

图/表 10

表1 PCR引物信息

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

图1 SLA-2-YT/pCDH 重组表达质粒示意图

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

图2 烟台黑猪SLA-2-YT基因CDS区PCR扩增与加尾 A 为烟台黑猪SLA-2-YT基因CDS区PCR扩增产物的检测图,M:DNA marker 2000;1:SLA-2-YT 基因CDS区的PCR扩增产物;B为PCR 扩增产物加Poly A尾后的回收产物检测图,M:DNA marker 2000;1:PCR 扩增产物经加尾后的回收产物

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

图3 重组质粒SLA-2-YT/pMD19-T Simple双酶切鉴定 M: DNA marker 2000, 1-7: 重组质粒SLA-2-YT/pMD19-T Simple双酶切产物

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

图4 Vector NTI 11.5分析SLA-2-YT/pMD 19-T Simple重组克隆载体中插入序列

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

图5 pCDH-CMV-MCS-EF1-Puro 载体回收 M:DNA marker 10000, 1:pCDH-CMV-MCS-EF1-Puro载体经Xba I和Not I 双酶切后的回收产物

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

图6 重组质粒SLA-2-YT/pCDH双酶切鉴定 M:DNA marker 10000, 1-3:重组质粒SLA-2-YT/pCDH双酶切产物

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

图7 嘌呤霉素作用于sT2细胞的致死浓度曲线 X轴为嘌呤霉素作用的浓度,Y轴不同浓度对应孔板中活细胞的数量

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

图8 光学显微镜下观察慢病毒感染后的细胞状态 A为PK15细胞,B为sT2细胞,C为转染空载体pCDH-CMV-MCS-EF1-Puro的sT2细胞,D为转染SLA-2-YT/pCDH的sT2细胞

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

图9 Western Blotting 检测 SLA-2-YT/pCDH 在sT2细胞中的表达 A 为Western Blotting检测结果;B为相对IOD值统计分析结果,以GAPDH（36 kD）作为内参衡量SLA-2-YT/pCDH蛋白（45 kD）的表达水平（**表示 P<0.01）

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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