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  

Preparation of polyclonal antibody against the histidine triad protein of Streptococcus suis and establishment of a dot-ELISA detection method

作　　者：蔡旭燊 燕书豪 赵芷若 郑峰 胡丹 卢亚维 申雪娇 何铠 熊晓辉[1] 操敏 卢一辰 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(College of Food Science and Light Industry,Nanjing Tech University,Nanjing 210035,China;Center for Disease Control and Prevention of Eastern Theater Command,Nanjing 210002,China)

机构地区：[1]南京工业大学食品与轻工学院,南京210035 [2]东部战区疾病预防与控制中心,南京210002

出　　处：《中国人兽共患病学报》2022年第8期657-665,共9页Chinese Journal of Zoonoses

基　　金：国家重点研发计划(No.2018YFC1602800)。

摘　　要：目的建立一种猪链球菌的快速、简便检测方法。方法根据猪链球菌2型强毒株05ZYH33组氨酸三联体蛋白HtpsC蛋白基因序列设计特异性引物,将HtpsC蛋白基因克隆至原核表达载体pET-30a并转化至大肠杆菌BL21(DE3),经0.2 mmol/L IPTG、15℃条件下诱导表达HtpsC蛋白。经Ni-IDA纯化后免疫新西兰白兔获得抗血清,通过亲和层析纯化制备Anti-HtpsC多克隆抗体。在此基础上,建立检测猪链球菌的斑点酶联免疫吸附检验法(Dot-ELISA),进一步优化了检测条件,并对该方法的特异性和敏感性进行评价。结果通过原核表达成功制备了HtpsC蛋白。Western blot结果显示,制备的Anti-HtpsC多克隆抗体可特异性结合HtpsC蛋白。优化的最佳多抗使用浓度为2.5μg/mL,最佳酶标二抗稀释倍数为1∶3000。特异性试验结果表明,含HtpsC蛋白基因的多种血清型猪链球菌均可呈现清晰的黄褐色斑点,但不含该蛋白基因的9型猪链球菌除外。而其他对照菌株:大肠杆菌、沙门氏菌、金黄色葡萄球菌、单增李斯特菌、化脓性链球菌、无乳链球菌和粪肠球菌均无斑点出现。灵敏度试验结果表明,该方法的检测限为1×10^(6)CFU/mL。结论本研究建立的Dot-ELISA方法特异性强,灵敏度良好,可用于检测多种血清型猪链球菌。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 2.5μg/mL,and the optimal dilution factor for the goat anti-rabbit IgG H&L(HRP)was 1∶3000.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 1×10^(6)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.

关 键 词：猪链球菌 HtpsC蛋白 DOT-ELISA 

分 类 号：S858.28[农业科学—临床兽医学]