柯萨奇病毒A16型VP1亚单位疫苗构建

The Construction of Coxsackievirus A16VP1 Subunit Vaccine

柯萨奇病毒A16型(coxsackievirus A16,CVA16)属于小RNA病毒科肠道病毒属,是手足口病(hand foot and mouth disease,HFMD)的主要病原体之一。近年来发现,CVA16感染的病患可引起脑部、肺部和心脏等的继发感染,甚至还可能引发肺炎、心肌炎和难治性休克等致死性并发症。大肠杆菌不耐热肠毒素B亚单位(Escherichia coli heat-labile enterotoxin B subunit,LTB),可作为特异性抗原的黏膜佐剂,用以增强特异性抗原的血清IgG抗体应答。大肠杆菌Ⅱ型不耐热肠毒素B亚单位(Escherichia coli type-Ⅱheat-labile enterotoxin B subunit,LT2B)具有比LTB更强的佐剂活性。本实验对CVA16VP1与LT2B融合蛋白作为抗原的免疫原性的研究,将初步评价产生的抗体的抗体效价,为进一步研究柯萨奇病毒的新型疫苗奠定基础。本研究构建pET32-CVA16VP1-LT2B融合表达载体,诱导表达CVA16VP1-LT2B蛋白,用于免疫小鼠,采取血清,检测特异性抗体效价。结果表明,本研究成功构建了pET32-CVA16VP1-LT2B融合表达载体,并成功提取到纯化的CVA16VP1-LT2B蛋白,间接ELISA检测出抗体效价为1∶800。

Coxsackievirus A16(CVA16)belongs to the enterovirus genus Picornaviridae and is one of the major pathogens of hand foot and mouth disease(HFMD).In recent years,it has been found that CVA16-infected patients can cause secondary infections such as brain,lung,and heart,and may even cause fatal complications such as pneumonia,myocarditis,and refractory shock.The Escherichia coli heat-labile enterotoxin B subunit(LTB)can be used as a mucosal adjuvant for specific antigens to enhance serum IgG antibody responses against specific antigens.The Escherichia coli type-Ⅱheat-labile enterotoxin B subunit(LT2B)has stronger adjuvant activity than LTB.We expressed the immunogenicity of CVA16VP1 and LT2B fusion proteins as antigens,and we will initially evaluate the antibody titers produced by the antibodies,which will lay the foundation for the further study of the novel vaccine against Coxsackie virus.We constructed a pET32-CVA16VP1-LT2B fusion expression vector to induce the expression of CVA16VP1-LT2B protein,which was used to immunize mice,take serum,and detect specific antibody titers.As a result,we successfully constructed a pET32-CVA16VP1-LT2B fusion expression vector and successfully extracted the purified CVA16VP1-LT2B protein.The indirect ELISA detected an antibody titer of 1∶800.