杆状病毒表面展示鸭坦布苏病毒E蛋白的免疫原性及保护效果的研究

Evaluation of duck Tembusu virus E protein based on baculovirus suface-display technology

鸭坦布苏病毒(DTMUV)为新出现的病毒,主要引起鸭的产蛋量急剧下降。为评价真核表达DTMUV的E蛋白的免疫原性,本研究利用杆状病毒表面展示技术,构建表面展示DTMUV JSXZ株E蛋白的重组杆状病毒(r BV-DTMUV-E),将其感染昆虫细胞,从而将E蛋白展示在杆状病毒的表面,并对表达的重组E蛋白(r E)的免疫原性和保护效果进行研究。经western blot和间接免疫荧光试验证明r E能够在Sf9细胞中有效表达;并且呈可溶性表达。重组蛋白经镍柱纯化,并采用氢氧化铝乳化(0.1μg/m L),分别以0.2 m L、0.4 m L和0.6 m L的剂量免疫雏鸭,通过ELISA抗体检测结果证明,r E可以诱导产生高水平的Ig G血清抗体;MTT试验结果表明,r E能够刺激T淋巴细胞增殖;攻毒保护试验结果显示,其0.2 m L组免疫保护率为80%,0.4 m L和0.6 m L组保护率均为100%,而对照组雏鸭均表现为典型的DTMUV感染症状,其中4只死亡。以上研究结果为DTMUV亚单位疫苗的研究奠定了基础。

Duck Tembusu virus （DTMUV） was newly emerged virus which causes severe egg-drop syndrome in duck.In the study,a recombinant baculovirus （rBV-DTMUV-E） contained E gene of DTMUV JSXZ stain was constructed by baculovirus surface-display technology,and the recombinant E protein（rE） was expressed and evaluated for the immunoprotection in ducklings.Western blot and indirect immunofluorescence assay showed that the rE protein was effectively expressed in rBV-DTMUV-E infected Sf9 cells in a soluble form.Subsequently,the rE was purified with nickel-chelate resin and emulsified with with aluminium hydroxide to immunize ducklings at a dosage of 0.1,ELISA detection results showed the high level of antibody was induced in the sera from the rE protein immunized ducklings.MTT results showed that rE protein was able to stimulate the proliferation ofT lymphocytes.Moreover,the protection rates of the rE protein were 75％ in 0.2 mL group and 100％ in both 0.4 mL and 0.6 mL groups,respectively,whereas,all duckings in control group shown the typical clinical signs with 4 died postchallenge.These results provided a basis for further study on DTMUV subunit vaccine.