A组牛轮状病毒基因工程亚单位疫苗的初步研究

Genetic engineering subunit vaccine of group A bovine rotavirus

目的构建轮状病毒外膜蛋白VP4、VP7基因与大肠杆菌LTB蛋白基因的重组表达载体,研究重组表达蛋白的免疫原性,为研制犊牛腹泻基因工程亚单位疫苗奠定基础。方法克隆A组牛轮状病毒临床分离株CHLY的VP4、VP7基因和大肠杆菌LTB基因,插入pET32a中构建了4个原核表达载体pET32a/VP7、pET32a/VP4、pET32a/VP7-LTB、pET32a/VP4-LTB,转化到BL21(DE3)中进行表达纯化,纯化蛋白与弗氏佐剂混合免疫10 w龄雌鼠,在实验期间对雌鼠进行交配,对其所产乳鼠用150μL10-6.5/0.1 mL TCID50的轮状病毒进行攻毒以研究重组蛋白免疫原性和攻毒保护性。结果重组蛋白在大肠杆菌BL21(DE3)中以包涵体的形式存在,Bandscan扫描分析结果显示,它们分别占菌体总蛋白的35%、20%、30%、18%,利用Ni-NTA Agarose在变性条件下成功纯化得到高纯度重组蛋白。10 w龄雌鼠免疫试验表明rVP4-LTB组激发了最强的免疫反应,rVP4组次之,rVP7-LTB组略高于rVP7,各个免疫组间的差异性不显著(P>0.05),各实验组IgG抗体水平显著高于空质粒对照组(P<0.01)。雌鼠所产乳鼠的攻毒实验表明,免疫组雌鼠所产乳鼠腹泻率为13.35%～23.8%,腹泻持续时间为48h～72h,较空质粒对照组明显降低,腹泻症状较轻且症状持续时间明显缩短。四个重组蛋白组攻毒保护率为76.2%～86.7%,rVP7-LTBr、VP4-LTB两组略高于rVP7r、VP4组,但相互间差异不显著(P>0.05)。结论重组蛋白具有较好的免疫原性,被免疫雌鼠体内产生的中和性抗体可以经被动免疫过程传递至子代,并为仔鼠提供保护。本研究为犊牛轮状病毒基因工程疫苗的研究奠定了一定的基础。

In order to acquire rotavirus outer membrane VP4, VP7 and LTB recombinant expression fusion protein and investigate its antigenicity and feasibility as an engineering subunit vaccine of group A bovine rotavirus against calf diarrhea, VP4 and VP7 gene of rotavirus isolate CHLY and LTB from E. coli was cloned and four prokaryotic recombinant expression vectors （pET32a/VP7, pET32a/VP4, pET32a/VPT-LTB and pET32a/VP4-LTB） were constructed and expressed. The recombinant proteins were purified and vaccines were prepared by mixing the protein with Freund＇s adjuvant. Antigenicity and challenge protection assay of four recombinant proteins were analyzed by immunizing the vaccines on 10w female mice whom were raised with male mice, and the suckling mice whom giving birth to were challenge with 150ul 10^-6.5/0. l mL TCID50 CHLY isolates. BandScan software analysis result showed that the ratio of rVP7, rVP4, rVP7-LTB and rVP4-LTB to total protein expressed by BL21 （DE3） were 35 %, 20 %, 30 % and 18 %, respectively. Statistical analysis of ten week female mice after immunity revealed that IgG antibody tilter of rVP4-LTB group was highest in the four group. The second one was rVP4 group,the third was rVP7 LTB group, and the forth was rVP7 group. The difference among the four experimental groups were not significant （P〉0.05）, but the difference between experimental group and control group was most significant （P〈0.01）. Challenging experiment of suckling mice showed that diarrhea ratio of immune group was 13.35%-23.8%, diarrhea duration time was 48h-72h, which was lower than control group. The diarrhea ratio was 100% and diarrhea duration time was 96h. Challenge protective ratio of four recombinant protein groups were 76.2%-86.7%, among which rVPT-LTB and rVP4-LTB group were a little higher than rVP7 and rVP4 group, but the difference was not significant （P〉0.05）. The experiment illustrated that the four recombinant proteins had better immunogenicity and the neutralization antibody of immune female mice could pass to its offspring and provide protection against rotavirus.