Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors

doi:10.3785/j.issn.1008-9209.2022.11.071

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (6): 873-880 DOI: 10.3785/j.issn.1008-9209.2022.11.071

Animal sciences & veterinary medicines

Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors

Mengke FENG^1,²(

),Xingbo WANG^1,²,Lulu LIN^1,²,Mingxian CUI^1,²,Yan YAN^1,²,Jiyong ZHOU^1,²(

)

^1.Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China
^2.Key Laboratory of Animal Virology of Ministry of Agriculture and Rural Affairs, Hangzhou 310058, Zhejiang, China

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Abstract

The aim of this study was to systematically investigate the differences in the soluble expression level of the African swine fever virus (ASFV) CD2v protein by different prokaryotic expression vectors, and the immunoreactivities of the inclusion body and soluble CD2v proteins were compared using clinical anti-ASFV antibody-positive sera. Five prokaryotic expression vectors, namely, pCold-TF, pET28a, pMAL-C6T, pGEX-4T-1 and pET32a, were utilized to express the CD2v protein without the signal peptide and transmembrane region, respectively. The inclusion body CD2v protein expressed by the pET28a vector and the soluble CD2v protein expressed by the pCold-TF vector were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and the immunoreactivity of the purified proteins was detected by indirect enzyme-linked immunosorbent assay (ELISA). The results showed that the CD2v protein expressed by the pCold-TF vector was soluble mainly, while the CD2v protein expressed by the pMAL-C6T vector was insoluble (inclusion body) and soluble, and the CD2v protein expressed by the other vectors was mainly as inclusion body. The indirect ELISA results for clinical anti-ASFV antibody-positive sera showed that the immunoreactivity of soluble protein was significantly better than that of the inclusion body protein (P＜0.05). The trigger factor (TF) tag of pCold-TF promoted the soluble expression of the CD2v protein, and the immunoreactivity of the expressed protein was greater than that of the inclusion body protein. This study lays the foundation for further immunogenicity research on the CD2v protein and provides a candidate strategy for the soluble expression of other important antigens.

Key words： African swine fever virus CD2v protein prokaryotic expression soluble expression immunoreactivity

Received: 07 November 2022 Published: 25 December 2023

CLC:

S855.3

Corresponding Authors: Jiyong ZHOU E-mail: mengkefeng@zju.edu.cn;jyzhou@zju.edu.cn

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Cite this article:

Mengke FENG,Xingbo WANG,Lulu LIN,Mingxian CUI,Yan YAN,Jiyong ZHOU. Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 873-880.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.11.071 OR https://www.zjujournals.com/agr/Y2023/V49/I6/873

不同原核表达载体对非洲猪瘟病毒CD2v蛋白可溶性表达及免疫反应性比较

本研究旨在系统性地探究不同原核表达载体对非洲猪瘟病毒CD2v蛋白的可溶性表达差别，并利用临床非洲猪瘟病毒抗体阳性血清比较包涵体和可溶性CD2v蛋白的免疫反应性。利用5种原核表达载体pCold-TF、pET28a、pMAL-C6T、pGEX-4T-1、pET32a分别表达去除信号肽和跨膜区的非洲猪瘟病毒CD2v蛋白，利用镍-氨三乙酸（nickel-nitrilotriacetic acid, Ni-NTA）亲和层析法分别纯化源自pET28a和pCold-TF表达载体的包涵体和可溶性CD2v蛋白，并用间接酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）方法比较纯化蛋白的免疫反应性。结果显示，pCold-TF载体以表达可溶性蛋白为主，pMAL-C6T载体同时表达包涵体和可溶性蛋白，而其他载体主要表达包涵体蛋白。临床非洲猪瘟病毒抗体阳性血清检测数据显示，可溶性蛋白的免疫反应性显著优于包涵体蛋白（P＜0.05）。pCold-TF载体的触发因子（trigger factor, TF）标签可促进CD2v蛋白的可溶性表达，其表达蛋白的免疫反应性优于包涵体蛋白。本研究结果为CD2v蛋白的免疫原性研究奠定了基础，亦为其他重要抗原的可溶性表达提供了思路。

关键词： 非洲猪瘟病毒, CD2v蛋白, 原核表达, 可溶性表达, 免疫反应性

Table 1 Different prokaryotic expression vector tags and molecular weights of recombinant proteins

Fig. 1 PCR identification results of recombinant plasmidsM: DNA marker; 1-5: Amplification products of pET-28a-CD2vΔSP-TM, pET32a-CD2vΔSP-TM, pGEX-4T-1-CD2vΔSP-TM, pMAL-C6T-CD2vΔSP-TM and pCold-TF-CD2vΔSP-TM recombinant plasmids, respectively; 6: Negative control.

Fig. 2 SDS-PAGE analysis of CD2v recombinant protein expression by five different prokaryotic expression vectorsA-E. pET28a-CD2vΔSP-TM, pET32a-CD2vΔSP-TM, pGEX-4T-1-CD2vΔSP-TM, pMAL-C6T-CD2vΔSP-TM and pCold-TF-CD2vΔSP-TM recombinant plasmids, respectively. M: Relative molecular weight of protein; 1: Uninduced bacteria; 2: Induced bacteria; 3: Bacterial supernatant by ultrasonic lysis after induction; 4: Bacterial precipitation by ultrasonic lysis after induction.

Fig. 3 Purification results of CD2v-TF recombinant proteinA. SDS-PAGE analysis; B. Western blotting analysis. M: Relative molecular weight of protein; 1-3: Heterologous proteins eluted with 20 mmol/L imidazole; 4-6: Target protein CD2v-TF eluted with 50 mmol/L imidazole; 7: Purified target protein CD2v-TF; 8: Induced bacteria; 9: Uninduced bacteria (control).

Fig. 4 Purification results of CD2v-His recombinant proteinA. SDS-PAGE analysis; B. Western blotting analysis. M: Relative molecular weight of protein; 1, 7: Uninduced bacteria (control); 2, 6: Induced bacteria; 3: Eluted heterologous protein; 4: Eluted target protein CD2v-His; 5: Purified target protein CD2v-His.

Fig. 5 Immunoreactivity of two CD2v recombinant proteins detected by indirect ELISASingle asterisk (*) indicates significant differences compared with the CD2v-His recombinant protein group at the 0.05 probability level, and n=3.


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