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目的构建羊布氏菌P39基因重组真核表达质粒,并检测其免疫效果。方法将前期克隆的P39基因片段连接到真核表达载体pcDNA3.1(+)的多克隆位点中,构建重组真核表达质粒pcDNA3.1-P39,转化E.coli DH5α,筛选阳性克隆,提取重组质粒,进行双酶切鉴定。以鉴定正确的重组质粒免疫BALB/c小鼠,凝集试验检测小鼠血清特异性抗体水平,ELISA法检测小鼠血清中细胞因子含量,MTT掺入法检测小鼠脾脏淋巴细胞的增殖效应。结果重组真核表达质粒pcDNA3.1-P39经双酶切鉴定构建正确;该重组质粒能够刺激小鼠产生特异性抗体(抗体峰值滴度为1∶320),显著增加Th1型细胞因子的产生,并能够增强小鼠脾脏淋巴细胞的增殖反应。结论成功构建了羊布氏菌P39基因重组真核表达质粒pcDNA3.1-P39,该重组质粒能够诱导BALB/c小鼠产生特异性免疫应答。
Objective To construct the recombinant eukaryotic expression plasmid of P39 gene of sheep and to test its immune effect. Methods The cloned P39 gene fragment was ligated into the multiple cloning site of eukaryotic expression vector pcDNA3.1 (+). The recombinant eukaryotic expression plasmid pcDNA3.1-P39 was constructed and transformed into E. coli DH5α. The positive clones were screened and extracted Recombinant plasmids, double enzyme digestion identification. The specific recombinant plasmids were used to immunize BALB / c mice. The levels of serum specific antibodies were detected by agglutination test. The levels of cytokines in serum were detected by ELISA. The proliferation of mouse splenic lymphocytes was detected by MTT assay. Results Recombinant eukaryotic expression plasmid pcDNA3.1-P39 was identified by double enzyme digestion. The recombinant plasmid can stimulate specific antibody (peak titer of antibody is 1: 320), significantly increase the production of Th1 type cytokines, And can enhance the mouse spleen lymphocyte proliferation reaction. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-P39 of P39 gene of B. bovine was successfully constructed. The recombinant plasmid can induce the specific immune response in BALB / c mice.