EgM123抗血清对体外培养原头蚴免疫杀伤研究

发布时间：2018-06-01 12:03

本文选题：原头蚴 + EgM123融合蛋白　；参考：《新疆农业大学》2015年硕士论文

【摘要】：棘球蚴病是由棘球绦虫中绦期(幼虫)引起的一种人畜共患寄生虫病,其中间宿主种类繁多,但绵羊是最适宜的中间宿主,犬是适宜终末宿主。包虫病防控措施可通过抑制终末宿主体内棘球绦虫成虫虫卵产生以切断病原循环链,从而减少棘球蚴病的传播。该寄生虫在中间宿主体内发育过程中,幼虫阶段产生的原头蚴(protoscolex,PSC),原头蚴在体外培养的条件下,可发育成囊泡,为防治包虫病在分子调控、药物干预等研究提供一个良好的模型。本研究主要是利用细粒棘球绦虫发育成熟的成虫体内与发育相关的基因Egm123,将其重组到质粒pET41b中并转染至大肠杆菌,经诱导表达、纯化GST-EgM123融合蛋白,并鉴定该蛋白的免疫活性,SDS-PAGE结果显示表达融合蛋白相对分子约为56.1 kD。用GST-EgM123融合蛋白免疫新西兰大白兔,制备高免血清,经Western blot鉴定表达蛋白的抗原性,并用ELISA方法测定抗体的效价,其效价达到1:320000以上。通过原头蚴体外培养技术检测EgM123特异性抗血清对其杀伤率,加入新鲜豚鼠血清开展含补体条件下特异性抗体对体外培养原头蚴的杀伤率。将原头蚴与GST-EgM123高免血清、His-EgM123高免血清和两种高免血清+豚鼠血清共培养,经相同培养时间和血清含量不等的培养条件下,与对照组观察高免血清在体外对原头蚴免疫杀伤作用。结果显示,在高免血清组中两种血清均灭活补体后,与对照组相比,原头蚴死亡率差异不显著(P0.05);正常血清组在200μL高免血清作用6天,原头蚴死亡率分别为42.53%和51.80%,与对照组相比,差异显著(P0.05);加入正常豚鼠血清(含补体)的高免血清组,与对照组相比,原头蚴死亡率达100%,差异显著(P0.05)。以上结果说明,制备出的EgM123抗血清,对体外培养的原头蚴具有免疫杀伤作用,为研制包虫病疫苗及进行相关诊断奠定了基础。
[Abstract]:Echinococcosis is a zoonotic parasitic disease caused by taeniasis (larva) of Echinococcus spp. There are many kinds of intermediate hosts, but sheep is the most suitable intermediate host and dog is the suitable terminal host. The prevention and control of echinococcosis can reduce the transmission of echinococcosis by inhibiting the generation of adult eggs of Echinococcus solium in the terminal host. During the development of the parasite in the intermediate host, the protoscolexus PSCS produced in the larval stage can develop into vesicles under the condition of culture in vitro, which provides a good model for the molecular regulation and drug intervention of hydatid disease control. In this study, Echinococcus granulosus (Echinococcus granulosus) mature adult Echinococcus granulosus (Echinococcus granulosus) was used to express and purify GST-EgM123 fusion protein by recombinant into plasmid pET41b and transfected into Escherichia coli. The results of SDS-PAGE showed that the relative molecule of the fusion protein was about 56.1 KD. The GST-EgM123 fusion protein was used to immunize New Zealand white rabbits to prepare high immune serum. The antigenicity of the expressed protein was identified by Western blot. The titer of antibody was determined by ELISA method, and the titer was over 1: 320 000. The killing rate of EgM123 specific antiserum was detected by means of culture technique in vitro, and the killing rate of specific antibody against protocercaria in vitro was carried out by adding fresh guinea pig serum. The hyperimmunized serum His-EgM123 of GST-EgM123 and two kinds of serum of guinea pig were co-cultured. Under the same culture time and different content of serum, the immune killing effect of hyperimmunized serum on procaria in vitro was observed with the control group. The results showed that there was no significant difference in the mortality of protocercaria between the two serum groups after inactivation of complement in the high immunized serum group compared with the control group, while in the normal serum group, the mortality rate was 42.53% and 51.80% after 6 days of exposure to 200 渭 L high immunized serum, respectively, compared with the control group, the mortality of the two groups was 42.53% and 51.80% respectively, compared with the control group. Compared with the control group, the mortality rate of protocephalosis reached 100, and the difference was significant (P0.05) in the high immunized serum group with normal guinea pig serum (including complement). The above results suggest that the antiserum prepared from EgM123 has the ability to kill the protocariae cultured in vitro, which lays a foundation for the development of hydatid vaccine and the diagnosis of hydatid disease.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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