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目的原核表达草鱼呼肠孤病毒(GCRV)HZ08株S6基因特定保守区域编码的外衣壳蛋白VP4并探究其免疫原性,为草鱼出血病(GCHD)防治提供新思路。方法将VP4基因特定保守区域克隆到原核表达质粒p ET-32a(+)中,诱导表达并用镍柱进行纯化,用纯化的目的蛋白免疫SD大鼠获取免疫血清;应用ELISA检测实验组(重组蛋白皮下免疫大鼠)和对照组(PBS免疫大鼠)血清Ig G抗体滴度及亚类的水平;应用间接免疫荧光实验(IFA)检测r VP4抗血清对病毒的识别情况。结果PCR、双酶切及DNA测序结果均表明p ET32a-VP4重组质粒构建成功;SDSPAGE结果表明目的基因在大肠杆菌BL21/DE3中获得高效表达,融合蛋白分子量约为43 k Da,且在37℃、1mmol/L IPTG条件下诱导表达效果最优;经亲和层析获得了高纯度蛋白;免疫大鼠6周后血清Ig G抗体滴度高达1∶819 200,Ig G1、Ig G2a水平差异无统计学意义(P>0.05);IFA结果显示抗血清组特异性荧光显著,而对照组未见荧光。结论 GCRV-HZ08 VP4具有良好的免疫原性,可诱导大鼠产生Th1/Th2混合型免疫反应,且r VP4抗血清可识别该病毒,为VP4作为疫苗候选分子提供了依据。
OBJECTIVE To prokaryotically express the coat protein VP4 encoded by the conserved region of S6 gene of grass carp reovirus (GCRV) HZ08 and explore its immunogenicity, providing new ideas for the prevention and control of grass carp hemorrhage (GCHD). Methods The specific conserved region of VP4 gene was cloned into the prokaryotic expression plasmid p ET-32a (+), induced to express and purified by nickel column, and then the immune serum was obtained by immunizing SD rats with the purified protein of interest. The expression of recombinant protein Subcutaneous immunized rats) and control group (PBS immunized rats) serum Ig G antibody titer and subclass level; indirect immunofluorescence assay (IFA) detection of r VP4 antisera to recognize the virus. Results The results of PCR, double enzyme digestion and DNA sequencing showed that the recombinant plasmid p ET32a-VP4 was successfully constructed. SDSPAGE results showed that the target gene was highly expressed in E. coli BL21 / DE3. The molecular weight of fusion protein was about 43 kDa, , 1mmol / L IPTG under the conditions of induced expression of the best results obtained by affinity chromatography of high purity protein; immunized rats 6 weeks after the serum Ig G antibody titer up to 1:819 200, Ig G1, Ig G2a levels were no difference Statistical significance (P> 0.05); IFA results showed that the antiserum specific fluorescence was significant, while the control group no fluorescence. Conclusions GCRV-HZ08 VP4 has good immunogenicity and can induce Th1 / Th2 mixed immune response in rats, and r VP4 antiserum can recognize this virus and provide basis for VP4 as vaccine candidate molecule.