鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达

doi:10.3969/j.issn.1004-1524.2020.12.04

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (12): 2138-2146.DOI: 10.3969/j.issn.1004-1524.2020.12.04

鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达

李仙春¹(), 芦艳², 毛耀芳¹, 杨海峰¹, 余海山¹, 马永华¹^,^*(), 万学瑞¹^,^*()

1.甘肃农业大学动物医学院,甘肃兰州 730070
2.西北师范大学国有资产管理处,甘肃兰州 730070

收稿日期:2020-04-09 出版日期:2020-12-25 发布日期:2020-12-25
通讯作者: 马永华,万学瑞
作者简介:万学瑞,E-mail: wamxr622@163.com
*马永华,E-mail: mayh517@163.com;
李仙春(1994—),女,藏族,甘肃永登人,硕士研究生,主要从事寄生虫及其分子生物学研究。E-mail: 920932549@qq.com
基金资助:
甘肃省自然科学基金(18JR3RA167);甘肃农业大学学科建设专项基金(GUA-XKJS-2018-073);甘肃农业大学人才专项经费项目(2017RCZX-11);甘肃农业大学青年导师基金(GAU-QDFC-2020-11)

Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli

LI Xianchun¹(), LU Yan², MAO Yaofang¹, YANG Haifeng¹, YU Haishan¹, MA Yonghua1¹^,^*(), WAN Xuerui¹^,^*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. State-Owned Assets Management Office, Northwest Normal University, Lanzhou 730070, China

Received:2020-04-09 Online:2020-12-25 Published:2020-12-25
Contact: MA Yonghua1,WAN Xuerui

摘要/Abstract

摘要：

试验旨在构建鸡Prnp基因原核表达载体,并在大肠埃希菌中进行表达,为制备鸡朊蛋白单克隆抗体提供材料。根据GenBank已报道的鸡Prnp基因组序列和pET-28a质粒多克隆位点设计引物,以健康的鸡全血基因组DNA为材料,采用PCR的方法扩增鸡的Prnp基因,将目的基因片段与pET-28a载体连接,构建重组原核表达载体。重组菌转化到E. coli BL21(DE3)感受态细胞中,并用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)进行诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达的重组蛋白。结果表明,重组蛋白在诱导剂的终浓度为0.08 mmol·L^-1,16 ℃,220 r·min^-1诱导7 h,蛋白表达量最高。综上所述,本研究成功构建了pET-28a-ChPrnp重组表达菌株,为Prnp朊蛋白的结构、生理功能和致病机制研究提供方法。

关键词: 鸡, Prnp基因, 重组表达载体, 原核表达

Abstract:

In order to construct the prokaryotic expression vector of chicken Prnp gene and express it in Escherichia coli, and to provide materials for the preparation of monoclonal antibodies against chicken prion protein, primers were designed according to chicken Prnp genome sequence reported by GenBank and the polyclonal site of pET-28a plasmid. Using healthy chicken whole blood genomic DNA as material, the Prnp gene of chicken was amplified by PCR. The target gene fragment was connected with pET-28a vector to construct recombinant prokaryotic expression vector. The recombinant bacteria were transformed into E. coli BL21(DE3) competent cells and induced by isopropyl-β-D-thiogalactoside (IPTG). The expressed recombinant protein was identified by SDS-PAGE. The results showed that the expression of recombinant protein was the highest at the final inducer concentration of 0.08 mmol·L ^-1, 16 ℃, 220 r·min^-1 for 7 hours. To sum up, the recombinant expression strain of pET-28a-ChPrnp was successfully constructed in this study, which provides a method for the study of the structure, physiological function and pathogenic mechanism of Prnp prion protein.

Key words: chicken, Prnp gene, recombinant expression vector, prokaryotic expression

中图分类号:

S831

李仙春, 芦艳, 毛耀芳, 杨海峰, 余海山, 马永华, 万学瑞. 鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达[J]. 浙江农业学报, 2020, 32(12): 2138-2146.

LI Xianchun, LU Yan, MAO Yaofang, YANG Haifeng, YU Haishan, MA Yonghua1, WAN Xuerui. Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli[J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2138-2146.

图/表 9

表1 连接体系

Table 1 Connection system

项目 Item	体积Volume/μL
项目 Item	1	2	3
pET-28a质粒片段pET-28a plasmid fragment	1.0	2.0	1.5
目的DNA Target DNA	7.0	6.0	6.5
10×连接酶缓冲液10×Ligase buffer	1.0	1.0	1.0
T4 DNA连接酶T4 DNA ligase	1.0	1.0	1.0
总体积Total volume	10	10	10

表2 蛋白胶配方

Table 2 Protein glue formula

试剂 Reagent	分离胶 Separating gel (12%)	浓缩胶 Concentrated gel(5%)
灭菌蒸馏水ddH₂O	3.3 mL	4.4 mL
30% ACR	4.0 mL	1.0 mL
1.5 mol·L^-1 Tris-HCl	2.5 mL	—
1.0 mol·L^-1 Tris-HCl	—	0.75 mL
10% SDS	0.1 mL	0.06 mL
10% APS	0.1 mL	0.06 mL
TEMED	10 μL	10 μL

图1 目的基因PCR扩增产物 M,DL2000 DNA marker;1,PCR扩增产物ChPrnp(822)。

Fig.1 PCR amplification product of target gene M, DL2000 DNA marker;1, PCR amplification product of ChPrnp(822).

图2 目的DNA与pET-28a质粒的酶切产物 M,DL5000 DNA marker;1,目的DNA酶切产物;2,pET-28a质粒酶切产物。

Fig.2 Digested product of target DNA and pET-28a plasmid M, DL5000 DNA marker; 1, Digested product of target DNA; 2, Digested product of pET-28a plasmid.

图3 重组表达质粒的菌液PCR鉴定 M,DL2000 DNA marker;1~3,1、2、3号菌液PCR扩增产物。

Fig.3 PCR identification of recombinant plasmid M, DL2000 DNA marker; 1-3, PCR amplification products of bacteria solution 1, 2 and 3.

图4 重组表达载体双酶切分析 M,DL5000 DNA marker;1~3,1、2、3号重组质粒双酶切产物。

Fig.4 Double enzyme digestion analysis of recombinant expression vector M, DL5000 DNA marker; 1-3, Double enzyme digestion products of recombinant plasmids 1, 2 and 3.

图5 重组菌诱导表达IPTG温度优化 1~3,16、28、37 ℃的温度下诱导表达的蛋白产物;M,蛋白质相对分子质量标准。

Fig.5 Temperature optimization of induced expression of IPTG by recombinant bacteria 1-3, Protein products induced at 16, 28 and 37 ℃; M, Protein marker.

图6 重组菌诱导表达IPTG浓度优化 1~5,分别加入IPTG的终浓度为0.08、0.09、0.10、0.11、0.12 mmol·L-1;M,蛋白质相对分子质量标准。

Fig.6 Optimization of concentration of IPTG induced by recombinant bacteria 1-5, Adding 0.08, 0.09, 0.10, 0.11, 0.12 mmol·L-1 IPTG, respectively; M, Protein marker.

图7 重组菌诱导表达时间优化 M,蛋白质相对分子质量标准;1~2,诱导5、7 h的重组菌表达产物;3,未诱导的重组菌。

Fig.7 Optimization of expression time of IPTG induced by recombinant bacteria M, Protein marker; 1-2, Expression products of recombinant bacteria induced for 5 and 7 h; 3, Uninduced recombinant bacteria.

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鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达

Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli

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