Preparation of polyclonal antibody against the histidine triad protein of Streptococcus suis and establishment of a dot-ELISA detection method
CAI Xu-shen1, YAN Shu-hao1, ZHAO Zhi-ruo1, ZHENG Feng2, HU Dan2, LU Ya-wei1, SHEN Xue-jiao1, HE Kai1, XIONG Xiao-hui1, CAO Min1, LU Yi-chen1
1. College of Food Science and Light Industry, Nanjing Tech University, Nanjing 210035, China; 2. Center for Disease Control and Prevention of Eastern Theater Command, Nanjing 210002, China
Abstract:In this study, a rapid and simple method for the detection of Streptococcus suis (S. suis) was established. Specific primers were designed on the basis of the HtpsC gene sequence of the histidine triad of the S. suis 2 virulent strain 05ZYH33. The HtpsC gene was cloned into the prokaryotic expression vector pet-30a and transformed into Escherichia coli BL21 (DE3). HtpsC protein was obtained by induction of expression at 15 ℃ with 0.2 mmol/L IPTG. Antiserum was obtained after immunization of New Zealand white rabbits with Ni IDA purified protein. Anti-HtpsC polyclonal antibodies were prepared through affinity chromatography purification. We established a dot enzyme-linked immunosorbent assay (dot-ELISA) for the detection of S. suis, and further optimized the assay conditions and evaluated the specificity and sensitivity of this method. We successfully prepared HtpsC protein through prokaryotic expression. Western blot results indicated that the prepared anti-HtpsC polyclonal antibody specifically bound HtpsC protein. The optimized optimal concentration of anti-HtpsC was 2.5 μg/mL, and the optimal dilution factor for the goat anti-rabbit IgG H&L (HRP) was 1:3 000. Specificity test results indicated that all serotypes of S. suis containing the HtpsC protein gene showed clear yellow brown spots, with the exception of S. suis 9, which did not contain the HtpsC gene. Other control strains (Escherichia coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis) showed no spots. The sensitivity test showed a limit of detection of 1×106 CFU/mL. Thus, a specific and sensitive dot ELISA method was established in this study, which can be used to detect most S. suis serotypes.
蔡旭燊, 燕书豪, 赵芷若, 郑峰, 胡丹, 卢亚维, 申雪娇, 何铠, 熊晓辉, 操敏, 卢一辰. 猪链球菌组氨酸三联体蛋白多抗的制备及Dot-ELISA检测方法的建立[J]. 中国人兽共患病学报, 2022, 38(8): 657-665.
CAI Xu-shen, YAN Shu-hao, ZHAO Zhi-ruo, ZHENG Feng, HU Dan, LU Ya-wei, SHEN Xue-jiao, HE Kai, XIONG Xiao-hui, CAO Min, LU Yi-chen. Preparation of polyclonal antibody against the histidine triad protein of Streptococcus suis and establishment of a dot-ELISA detection method. Chinese Journal of Zoonoses, 2022, 38(8): 657-665.
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