两种旋毛虫丝氨酸蛋白酶抑制剂对小鼠免疫保护效果的评估  

Evaluation of immunoprotective effects of two serine protease inhibitors ofTrichinella spiralis in mice

作　　者：甄晶博 刘家欣 林立浩 孙凤 张玉恒 王瑞彪 李志欣 韩阳[1] 刘照琨 路义鑫[1] ZHEN Jing-bo;LIU Jia-xin;LIN Li-hao;SUN Feng;ZHANG Yu-heng;WANG Rui-biao;LI Zhi-xin;HAN Yang;LIU Zhao-kun;LU Yi-xin(Heilongjiang Key Laboratory for Zoonosis,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学,动物源性人兽共患病黑龙江省重点实验室,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2023年第6期750-756,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31372427)。

摘　　要：为评估旋毛虫丝氨酸蛋白酶抑制剂对BALB/c小鼠的免疫保护作用,选用2种重组表达的Serpin型和Kazal型旋毛虫丝氨酸蛋白酶抑制剂(TsAdSPI和TsKaSPI),联合弗氏佐剂通过腹腔注射的方式单独免疫小鼠,用间接ELISA法检测免疫后小鼠特异性血清抗体IgG以及IgG亚型(IgG1和IgG2a)的水平,用CCK-8法测定重组蛋白刺激后脾淋巴细胞的增殖情况,用ELISA法检测脾淋巴细胞培养液上清中细胞因子IFN-γ、IL-10、IL-12和IL-4的水平;使用旋毛虫肌幼虫攻击感染免疫后的小鼠,计算并分析小鼠肠道成虫减虫率及肠道病理变化,并评估TsAdSPI和TsKaSPI与ISA206佐剂联合免疫小鼠后对小鼠肠道成虫减虫率的影响。结果表明,与PBS组相比,2种重组蛋白所诱导的IgG和IgG亚型抗体水平均明显升高,IL-12、IFN-γ、IL-4和IL-10表达水平也均显著升高,且重组蛋白可诱导免疫小鼠脾淋巴细胞的体外增殖;攻虫结果表明,2种重组蛋白免疫后小鼠肠道旋毛虫成虫数量较PBS组显著减少且肠道病理变化减轻;2种重组蛋白联合免疫组的成虫减虫率高于单独免疫组。上述结果表明,重组蛋白TsAdSPI和TsKaSPI均可诱导小鼠产生较强的体液免疫和细胞免疫应答,并且能够在一定程度上阻止旋毛虫肌幼虫对小鼠肠道的入侵,减轻旋毛虫对小鼠肠道的病理损伤,且TsAdSPI和TsKaSPI联合免疫降低旋毛虫入侵的效果更显著。In order to evaluate the immunoprotective effect of Trichinella spiralis serine protease inhibitors in BALB/c mice,two recombinant Serpin and Kazal serine protease inhibitors(TsAdSPI and TsKaSPI)were used to immunize mice by intraperitoneal injection with Freund’s adjuvant.The levels of specific serum antibody IgG and IgG subtypes(IgG1 and IgG2a)of immunized mice were measured by indirect ELISA,the proliferation of spleen lymphocytes stimulated by recombinant proteins was detected using CCK-8,and the levels of cytokines IFN-γ,IL-10,IL-12 and IL-4 in the supernatant of spleen lymphocyte culture were detected by ELISA.After immunization,mice were infected with T.spiralis muscle larvae,and the reduction rate of intestinal adult worms and intestinal pathological changes of mice were calculated and analyzed.And the immune effect of TsAdSPI and TsKaSPI combined with ISA206 adjuvant on the reduction rate of intestinal adult worms in mice was evaluated.The results indicated that the levels of IgG and IgG subtype antibodies induced by the two recombinant proteins were significantly higher than those in the PBS group,and the levels of IL-12,IFN-γ,IL-4,and IL-10 were also increased significantly,and the recombinant proteins can induce proliferation of spleen lymphocytes isolated from immunized mice in vitro.The results of T.spiralis infection indicated that the numbers of intestinal adult worms in mice immunized with the two recombinant proteins were significantly reduced and the intestinal pathological changes were alleviated compared with the PBS group.The adult worms reduction rate of combined immunization with two recombinant proteins was higher than that of individual immunization group.The above results indicated that the recombinant proteins TsAdSPI and TsKaSPI could induce strong humoral and cellular immune responses in mice,and prevent the intestinal invasion of T.spiralis muscle larvae to a certain extent,and reduce the pathological damage of T.spiralis to the intestine of mice.The combined immunization effect

关 键 词：旋毛虫 丝氨酸蛋白酶抑制剂 免疫保护 

分 类 号：S852.731[农业科学—基础兽医学]