两个对虾新型表达载体的构建

Constructing two novel shrimp expression vectors

【目的】构建一种能在对虾细胞内稳定存在的新型表达载体,为对虾口服疫苗的研制奠定基础。【方法】设计特异性引物,以对虾白斑综合症病毒(WSSV)DNA为模板,PCR扩增早期基因启动子IE1的不同长度片段IE(-94/+52)和IE(-945/+52);利用限制性酶切及连接的方法,以IE(-94/+52)和IE(-945/+52)替换pcDNA3.1-GFP的CMV启动子;然后将构建好的表达载体与阳离子脂质体转染试剂TransLipidTM混合,肌肉注射凡纳滨对虾。【结果】经双酶切鉴定,表达载体pcDNA3.1-IE(-94/+52)-GFP、pcDNA3.1-IE(-945/+52)-GFP分别获得6150和146 bp、6150和997 bp的目的条带;注射pcDNA3.1-IE(-94/+52)-GFP、pcDNA3.1-IE(-945/+52)-GFP的对虾部分体细胞在荧光显微镜下可见绿色荧光,且注射pcDNA3.1-IE(-94/+52)-GFP的绿色荧光强度强于注射pcDNA3.1-IE(-945/+52)-GFP。【结论】构建的两个新型表达载体pcDNA3.1-IE(-94/+52)-GFP、pcDNA3.1-IE(-945/+52)-GFP均可用于对虾病毒抗原蛋白基因的表达。

【Objective】In order to provide references for shrimp oral vaccine research, two novel shrimp expression vectors were constructed. 【Method】Using the WSSV genome DNA as the template, special primers were designed and WSSV early promoter IE1 fragment IE（-94/＋52） and IE（-945/＋52） were amplified. Novel vectors were constructed by replacing the CMV promoter of vector pcDNA3.1-GFP with IE（-94/＋52） and IE（-945/＋52） respectively via the restriction enzyme digestion and connection methods. The constructed vectors were injected to the tested Litopenaeus vannamei shrimps after mixing with cationic liposome TransLipidTM. 【Result】By restriction enzyme digestion vector 6150 bp and target fragments 146 and 997 bp which coincided with IE（-94/＋52）and IE（-945/＋52） were found respectively and then confirmed by sequencing. Animal experiment results showed that green fluorescence was observed by fluorescence microscope after shrimps were injected with pcDNA3.1-IE（-94/＋52）-GFP or pcDNA3.1-IE（-945/＋52）-GFP. The green fluorescence was more intense in the shrimps injected with pcDNA3.1-IE（-94/＋52）-GFP than with pcDNA3.1-IE（-945/＋52）-GFP. 【Conclusion】Novel shrimp expression vector pcDNA3.1-IE（-94/＋52）-GFP or pcDNA3.1-IE（-945/＋52）-GFP could be used for expressing prawn viral antigen protein gene.