Prokaryotic Expression of Recombinant Bovine Mastitis Staphylococcus aureus GapC Protein and Identification of Its B-cell Epitopes

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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

Fig. 5 Tertiary structure prediction of recombinant GapC

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

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

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

Fig. 8 Epitope identification results of recombinant GapC

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