猪链球菌重组靶向表位蛋白DCpep-GSE的构建及细胞毒性检测

doi:10.16431/j.cnki.1671-7236.2021.08.032

摘要/Abstract

摘要： 试验旨在构建pET28a-DCpep-GSE原核表达载体，并制备猪链球菌（Streptococcus suis）重组表位蛋白。应用生物信息学方法预测猪链球菌保护性抗原三磷酸甘油醛脱氢酶（GAPDH）、烯醇化酶（Enolase）、DNA核酸酶（SsnA）的跨膜区结构、信号肽、B细胞表位、辅助T细胞（Th细胞）表位及蛋白的二级结构；设计重组蛋白（DCpep-GSE蛋白）的氨基酸序列并分析其理论等电点、亲疏水性等理化性质；构建重组原核表达载体pET28a-DCpep-GSE，转化大肠杆菌BL21（DE3）感受态细胞，优化诱导表达条件，His镍柱纯化并检测目的蛋白的细胞毒性和溶血性。结果显示，SsnA蛋白第1-50及1 036-1 059位氨基酸为胞内或跨膜片段，第1-56位氨基酸为信号肽片段，GAPDH和Enolase无跨膜区及信号肽；B细胞和Th细胞表位处于二级结构中的无规则卷曲与β-转角区域，表位抗原性良好；DCpep-GSE蛋白共373个氨基酸，分子质量为45.3 ku，理论等电点（pI）为4.57，平均亲水性（GRAVY）为-0.677，属于酸性亲水性蛋白；质粒双酶切得到5 369 bp的载体条带和1 131 bp的目的条带，PCR扩增产物测序结果与设计序列一致，载体构建正确；以1 mmol/L IPTG于37 ℃诱导6 h蛋白表达量最高，超声破碎后得到可溶性蛋白；试验组蛋白浓度为500 μg/mL时，RAW264.7、PK15和MARC145细胞的相对增殖率分别达到92.3%、99.5%和99.7%，细胞形态与对照组一致，生长旺盛；各样品组的溶血率均<5%。本研究设计了重组蛋白DCpep-GSE，成功地表达和纯化了DCpep-GSE蛋白，并验证了其安全性，为后续猪链球菌病亚单位疫苗的研制奠定了基础。

关键词: 猪链球菌; 树突状细胞靶向肽; 抗原表位; 亚单位疫苗; 细胞毒性

Abstract: The aim of this study was to prepare recombinant epitope protein of Streptococcus suis (S.suis) by constructing prokaryotic expression vector pET28a-DCpep-GSE.The transmembrane structures, signal peptides, B cell epitopes, Th cell epitopes and secondary structures of GAPDH, Enolase and SsnA of S.suis were analyzed and predicted by bioinformatics.The amino acid sequence of the recombinant protein (DCpep-GSE protein) was designed and its theoretical isoelectric point, hydrophilicity and other physicochemical properties were analyzed.Recombinant prokaryotic expression vector pET28a-DCpep-GSE was constructed and transformed into E.coli BL21 (DE3).The induced expression conditions were optimized, and the target protein was purified by His nickel column and tested for cytotoxicity and hemolysis.The results showed that the amino acids at sites 1-50 and 1 036-1 059 of SsnA protein were intracellular or transmembrane segments, and the amino acids at sites 1-56 were signal peptides.GAPDH and Enolase had no transmembrane regions or signal peptides.B and Th cell epitopes were located in random coil and β-turn region of secondary structure, showed that epitopes had good antigenicity.DCpep-GSE protein contained 373 amino acids, with molecular weight of 45.3 ku, theoretical isoelectric point (pI) of 4.57, and GRAVY of -0.677, belonged to the category of acidic hydrophilic protein.A 5 369 bp vector band and a 1 131 bp target band were obtained by double digestion of the plasmid.The sequencing results of PCR amplification products were consistent with the designed sequence, and the vector construction was correct.The highest protein expression was induced by 1 mmol/L IPTG at 37 ℃ for 6 h, and the soluble protein was obtained after ultrasonic crushing.The relative proliferation rate of RAW264.7, PK15 and MARC145 cells were 92.3%, 99.5% and 99.7%, respectively, when the protein concentration was 500 μg/mL in experimental group.The cell morphology was the same as that in control group, and the cells grew well.The hemolysis rate of each sample group was less than 5%.In this study, the recombinant protein DCpep-GSE was designed by bioinformatics method, and then DCpep-GSE was successfully expressed and purified by prokaryotic expression system, and its safety was verified.It laid a foundation for the further development of subunit vaccine of S.suis.

Key words: Streptococcus suis; DC targeting peptide; antigenic epitopes; subunit vaccine; cytotoxicity

中图分类号:

S852.61

潘晨浩, 张欣, 赵瑞利, 姜轩, 金天明, 于恩远, 李留安, 曾君, 于晓雪, 宋淇淇, 胡烨. 猪链球菌重组靶向表位蛋白DCpep-GSE的构建及细胞毒性检测[J]. 中国畜牧兽医, 2021, 48(8): 2990-3001.

PAN Chenhao, ZHANG Xin, ZHAO Ruili, JIANG Xuan, JIN Tianming, YU Enyuan, LI Liuan, ZENG Jun, YU Xiaoxue, SONG Qiqi, HU Ye. Construction and Cytotoxicity Detection of Recombinant Epitope Protein DCpep-GSE of Streptococcus suis[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2990-3001.

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