重组牛乳源金黄色葡萄球菌GapC蛋白优势B细胞抗原表位的预测和筛选

doi:10.13560/j.cnki.biotech.bull.1985.2022-0962

摘要/Abstract

摘要：

旨在表达牛乳源金黄色葡萄球菌（Staphylococcus aureus）GapC蛋白并对其B细胞抗原表位进行预测与鉴定，本研究利用实验室分离鉴定的S. aureus分离株15119 扩增GapC基因并构建重组表达质粒pET-28a-GapC，诱导纯化得到分子量为44 kD重组蛋白GapC，以此免疫新西兰大白兔，获得特异多克隆抗体。利用生物信息学方法，对GapC蛋白的二级及三级结构进行分析，预测其B细胞抗原表位，并利用特异性抗体对筛选的表位进行鉴定。结果表明，GapC蛋白具有良好的免疫原性，筛选出7个线性B细胞抗原表位，利用兔抗重组GapC蛋白多克隆抗体鉴定得到了PL 5（²²¹IPEIDGKLDGGAQRVP²³⁶）多肽和PL 7（²⁶⁴KNASNESFGYTEDEIVSSDVVGM²⁸⁶）2个优势B细胞表位。本研究成功制备了GapC蛋白，预测并鉴定了2个优势抗原表位，为其嵌合表位疫苗的开发提供了技术支持。

关键词: 牛源金黄色葡萄球菌, GapC蛋白, 表达, B细胞抗原表位, 预测与鉴定

Abstract:

To express the GapC protein of Staphylococcus aureus isolated from bovine milk and to predict and identify its B-cell epitope, GapC gene of S. aureus strain 15119 isolated by our lab was amplified and the recombinant expression plasmid pET-28a-GapC was constructed. The recombinant protein GapC with a molecular weight of 44 kD was induced and purified. Then, the purified protein was used to immunize New Zealand white rabbits to obtain polyclonal antibodies. And bioinformatics method was to analyze the secondary and tertiary structures of GapC protein and predict the B cell epitopes. The predicted and selected epitopes were identified by specific antibodies. The results showed that the GapC protein had promising immugenicity and 7 B cell epitopes were selected. Two of dominant B cell epitope PL5（²²¹IPEIDGKLDGGAQR-VP²³⁶）and PL7（²⁶⁴KNASNESFGYTEDEIVSSDVVGM²⁸⁶）were identified using specific polyclonal antibody. This data lays the foundation for the development of epitope vaccine.

Key words: bovine Staphylococcus aureus, GapC protein, expression, B cell epitope, prediction and identification

陈晓萌, 张雪静, 张欢, 张宝江, 苏艳. 重组牛乳源金黄色葡萄球菌GapC蛋白优势B细胞抗原表位的预测和筛选[J]. 生物技术通报, 2023, 39(5): 306-313.

CHEN Xiao-meng, ZHANG Xue-jing, ZHANG Huan, ZHANG Bao-jiang, SU Yan. Prokaryotic Expression of Recombinant Bovine Mastitis Staphylococcus aureus GapC Protein and Identification of Its B-cell Epitopes[J]. Biotechnology Bulletin, 2023, 39(5): 306-313.

图/表 10

图1 GapC 基因的PCR扩增及重组质粒的双酶切鉴定 A：GapC 基因的PCR扩增；M1: DL 2000 DNA Marker;1-4: 扩增的GapC 基因; 5：阴性对照; B：GapC 基因重组质粒的双酶切鉴定；M2: DL 5000 DNA Marker; 1-4: pET-28a-GapC双酶切鉴定

Fig. 1 PCR amplification of GapC gene and double digestion of recombinant plasmid A：PCR amplification of GapC gene；M1:DL 2000 DNA Marker;1-4: amplification of GapC gene,5: negative control. B：Double digestion of recombinant plasmid of GapC gene; M2:DL 5000 DNA Marker;1-4: double digestion of pET-28a-GapC

图2 重组GapC蛋白的诱导及纯化结果 M: 蛋白Marker; 1: GapC未诱导; 2: GapC诱导后; 3:上清; 4 : 沉淀; 5:穿透; 6-7:洗脱

Fig. 2 Induction and purified result of recombinant GapC M: Protein marker; 1:uninduced by GapC; 2: after induced by GapC; 3: supernatant; 4: precipitation; 5: penetration; 6-7: elution

图3 重组GapC蛋白的Western blot分析 M: 蛋白预染Marker; 1:诱导后 GapC; 2: 未诱导 GapC

Fig. 3 Western blot analysis of recombinant protein GapC M: Prestained protein marker; 1: after induced by GapC; 2: uninduced by GapC

图4 重组GapC蛋白的信号肽与跨膜区分析 A：信号肽区；B：跨膜区域

Fig. 4 Analysis of signal peptide regions and transmembrane regions of GapC A:signal peptide regions; B:transmembrane regions

表1 重组GapC蛋白的二级结构预测

Table 1 Secondary structure prediction of recombinant GapC protein

二级结构 Secondary structure	占总氨基酸比例 Percentage in total amino acids/%
Hh: α-螺旋	31.07
Ee: β-片层	25.74
Tt: β-转角	9.47
Cc: 无规则卷曲	33.73

图5 重组GapC蛋白三级结构预测

Fig. 5 Tertiary structure prediction of recombinant GapC

表2 重组GapC蛋白抗原表位预测

Table 2 Prediction of antigen epitope for recombinant GapC protein

多肽 Polypeptide	多肽位置 Polypeptide position	多肽序列 Polypeptide sequence
PL1	66-90	RVNGKEVKSFSEPDASKLPWKDLNI
PL2	95-115	ECTGFYTDKDKAQAHIEAGAK
PL4	181-208	TGDQNTQDAPHRKGDKRRARAA AENIIP
PL5	221-236	IPEIDGKLDGGAQRVP
PL6	247-268	VVLEKQDVTVEQVNEAMKNASN
PL7	264-286	KNASNESFGYTEDEIVSSDVVGM
PC8	297-308	TRVMSVGDRQLV

图6 预测的B细胞抗原表位在GapC三级结构中的位置

Fig. 6 Position of predicted B cell epitope in GapC tertiary structure

图7 兔抗重组GapC抗血清抗体水平及滴度检测结果 A：兔抗重组GapC抗血清抗体水平检测结果；B：兔抗重组GapC抗血清抗体滴度检测结果，不同字母表示组间差异显著（P<0.05），下同

Fig. 7 Immunized rabbit serum antibody levels and titer detection results of recombinant GapC A：Immunized rabbit serum antibody levels detection results. B：Immunized rabbit serum antibody levels detection results, and different letters indicate significant differences between groups（P<0.05）, the same below

图8 重组GapC蛋白抗原表位的鉴定结果

Fig. 8 Epitope identification results of recombinant GapC

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