多杀性巴氏杆菌重组Dot蛋白对小鼠免疫保护效果研究  

Immune protective efficacy of recombinant Dot protein of Pasteurella multocida in mice

作　　者：王宇凤 苏静明 邵萌萌 刘鑫[1] 黄复深[1,2] WANG Yu-feng;SU Jing-ming;SHAO Meng-meng;LIU Xin;HUANG Fu-shen(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Engineering Reasearch Center of Veterinary Drug,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南农业大学湖南兽药工程技术研究中心,湖南长沙410128

出　　处：《中国预防兽医学报》2023年第11期1165-1171,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：湖南省自然科学基金(2017JJ2116)。

摘　　要：为评估猪多杀性巴氏杆菌(Pm)毒力蛋白Dot诱导小鼠产生的免疫保护效果,本研究根据GenBank登录的A型Pm CS株dot基因序列设计特异性引物,以CS株基因组DNA为模板经PCR扩增dot基因片段,PCR产物测序后利用生物信息学软件进行序列分析。结果显示,dot基因长729 bp,编码含242 aa的膜蛋白;该蛋白的N端有一个24 aa组成的信号肽,具有重复Sel 1结构功能域,属于四肽重复超家族;抗原表位分析显示,Pm Dot蛋白含有6个抗原表位;序列同源性分析显示,不同血清型Pm Dot蛋白氨基酸序列的同源性达99%以上。将dot基因克隆至原核表达载体pET-28a(+)中构建重组表达质粒pET-dot,转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白,采用His纯化柱纯化后,经SDS-PAGE检测并采用western blot鉴定。结果显示,重组Dot蛋白(rDot)正确表达,且纯化效果较好,具有较强的反应原性。取40只4周龄BALB/c小鼠随机均分为5组进行免疫保护实验,3个实验组分别于0、2周、4周对小鼠通过背部皮下多点注射弗氏不完全佐剂(IFA)+10μg rDot(低剂量组)、IFA+30μg rDot(中剂量组)、IFA+50μg rDot(高剂量组)。两个对照组分别注射等量生理盐水和IFA。共免疫3次,每次间隔2周,小鼠于免疫前和攻毒前,均经尾静脉采血并分离血清,采用ELISA检测血清中IgG抗体水平。第3次免疫后2周,通过腹腔注射10×LD_(50)的A型Pm CS株攻击小鼠,观察攻毒后小鼠的临床症状及死亡情况。结果显示,生理盐水和IFA组小鼠抗体水平无显著差异;实验组小鼠二免后2周和三免后2周IgG抗体水平均极显著高于两个对照组(P<0.01)。攻毒后3 d,对照组小鼠全部死亡,低剂量、中剂量、高剂量组小鼠存活率分别为12.5%(1/8)、50%(4/8)、62.5%(5/8)。攻毒后5 d存活小鼠逐渐恢复并正常采食。本研究表明,rDot具有很好的免疫原性,可诱导小鼠产生部分免疫保护力。该研究为Pm Dot亚单位疫苗的研究积累了基础资To evaluate its immune protective efficacy of the virulent Dot protein of Pasteurella multocida(Pm)in mice,a pair of specific primers were designed based on the dot gene sequence of Pm CS strain in GenBank,the dot gene fragment was amplified by PCR from genomic DNA of Pm serotype A CS strain.Sequencing and bioinformatics analysis of the products showed that the dot gene consisted of 729bp and encoded a membrane protein containing 242 amino acids.The N-terminus of this protein had a signal peptide composed of 24 amino acids,with a repeat sel 1 domain,belonging to the tetrapeptide repeat superfamily.Epitope analysis showed that the protein had 6 antigenic epitopes.Sequence homology analysis showed that it shared more than 99%homology with other Dot proteins from different serotypes Pm.The dot gene without signal peptide sequence regions was subcloned into the prokaryotic expression vector pET-28a(+)to construct the recombinant expression plasmid pET-dot,which was transformed into E.coli BL21(DE3).The recombinant Dot protein(rDot)induced by IPTG was purified by his-taaged purification column,and detected and identified by SDS-PAGE and western blot.The results showed that the rDot was expressed correctly,purified effectively,and had a good reactogenicity.Forty BALB/c mice of four-week-age were randomly divided into 5 groups for immune protection experiment,the mice in three experimental groups were subcutaneously injected with Freund's incomplete adjuvants(IFA)+10μg rDot(low-dose group),IFA+30μg rDot(medium-dose group),and IFA+50μg rDot(high-dose group)at 0,2 and 4 weeks,respectively.The mice in two control groups were injected with equal amounts of IFA and normal saline,respectively.Before each immunization and infection,blood was sampled from the tail vein and sera were separated,and the IgG levels in serum were detected by enzyme-linked immunosorbent assay(ELISA).Two weeks after the third immunization,the mice were intraperitoneally challenged with Pm serotype A CS strain at a dose of 10×LD_(50),and the clinic

关 键 词：多杀性巴氏杆菌 dot基因 重组Dot蛋白 免疫保护效果 小鼠 

分 类 号：S852.61[农业科学—基础兽医学]