凡纳滨对虾C-型凝集素基因的原核表达与免疫作用

Prokaryotic expression of C-type lectin gene of Litopenaeus vannamei and immunologic activity evaluation of recombinant protein

为研究凡纳滨对虾C-型凝集素的免疫功能,实验利用原核表达系统对凡纳滨对虾C-型凝集素基因的开放阅读框进行了重组表达,并通过纯化分离获得重组目的蛋白,采用血细胞悬浮培养的方法,研究了重组C-型凝集素对凡纳滨对虾血细胞免疫作用的影响。实验梯度设置为对照组和重组C-型凝集素蛋白(1.0 mg/mL),分别在0、3、6、9、12、24 h取培养的血细胞和培养液进行免疫相关指标的测定。结果表明,重组C-型凝集素经SDS-PAGE分析显示在39 ku处有显著诱导条带,与预测的分子量大小基本一致,Western-blotting分析可以与anti-His抗体特异性结合,从而证明C-型凝集素重组蛋白在大肠杆菌中成功表达。重组C-型凝集素对大肠杆菌具有明显的凝集作用,对凡纳滨对虾血细胞数量、存活率和吞噬率影响显著;培养基酚氧化酶活力在培养3～12 h内呈峰值变化,于9 h时达到最大值。研究表明,C-型凝集素不仅具有凝集作用,而且还能激活酚氧化酶原系统,具有重要的免疫防御作用。

The studies on innate immune responses of Litopenaeus vannamei C-type lectin, were conducted by recombinant C-type lectin protein through expression in Escherichia coli. We used cell suspension culture to study the effect of C-type lectin on the cellular immune response of Litopenaeus vannamei. The experiment was divided into two groups ： control group and recombinant protein （ 1.0 mg/mL）. After 0,3,6, 9,12,24 hours ,we harvested the solutions and then assessed parameters respectively. The results showed that SDS-PAGE revealed the molecular weight of LvLec protein was 39 ku, which was consistent with the predicted size. Western-blotting revealed it could bind anti-His antibody, which proved that C-type lectin recombinant protein in Escherichia coli succeeded in expression. For the hernagglutination assay, massive agglutination could be observed after Escherichia coli was incubated with LvLec for 1 h. At the same time, LvLec protein had a significant effect on haemocyte count, cell viability and phagocytic activity of white shrimp Litopenaeus vannamei（P 〈 0.05 ）. The extracelluar phenoloxidase activity of the LvLec protein group showed peak changes during 3 - 12 h, and at 9 h the extracelluar phenoloxidase activity was highest, then returned to the control level （ P 〈 0.05 ）. Therefore, crustacean C-type lectin not only had the function of hemagglutination,but also activated prophenoloxidase activating system, and probably played an important role in immune response.