以多层磷酸钙纳米颗粒为载体的O型口蹄疫病毒VP1和VP2基因重组疫苗的构建

Construction of a Recombinant VP1 and VP2 of Type O Foot-and-Mouth Disease Virus DNA Vaccine Coated with Multi-shell Calcium Phosphate Nanoparticles

构建以多层纳米颗粒为载体的O型口蹄疫病毒基因重组疫苗。在GenBank中查询编码O型口蹄疫病毒(FMDV)VP1 141-160氨基酸和VP2 1-33氨基酸的核苷酸序列,合成该两段基因的重组基因并命名为VP1-VP2基因。再通过PCR扩增VP1和VP2基因,并将VP1、VP2及VP1-VP2基因分别插入到真核表达载体pcDNA3.1中,构建3种重组真核表达质粒(pcDNA3.1-VP1、pcDNA3.1-VP2和pcDNA3.1-VP1-VP2),并分别对重组质粒进行酶切、PCR鉴定及测序分析。将构建的重组质粒制备成多层磷酸钙纳米颗粒,检测其粒径、稳定性及体外转染效率。重组质粒酶切结果显示,基因片段大小与预期相符;测序显示与GenBank中相应基因序列同源性为100%。制备的多层纳米颗粒粒径约为530nm;在4℃过夜保存后,溶液均匀透明;体外转染HEK293细胞的转染效率在9.6%左右。成功构建了以多层磷酸钙纳米颗粒为载体的O型口蹄疫病毒VP1和VP2基因重组质粒,为进一步研究该基因疫苗奠定了基础。

A recombinant gene vaccine containing VP1 and VP2 genes which encoding VP1 141-160 and VP2 1-33 aa of type O foot-and-mouth disease virus coated multi-shell calcium phosphate nanoparticles was constructed. The VP1 and VP2 genes encoding VP1 141-160 and VP2 1-33 aa of type O FMDV were syn- thesized and linked as recombinant VP1-VP2 gene, whose sequences came from the Genbank. Then the VP1 gene and VP2 gene were amplified by PCR from the recombinant VP1-VP2 gene. After that,the VP1 gene,VP2 gene and the recombinant VP1-VP2 gene were constructed separately into eukaryotic expression vector pcDNA3. 1 as recombinant plasmids pcDNA3. 1-VP1, pcDNA3. 1-VP2 and pcDNA3. 1-VPI-VP2. Then the recombinant plasmids were identified by enzymatic digestion, PCR and sequence analysis. At last,the plasmids DNA were encapsulated in multi-shell calcium phosphate nanoparticles. And the diameters, stability and transfection efficiency of these nanoparticles were detected in vitro. The enzymatic digestion and PCR assay of the recombinant plasmids indicated that the exogenous gene was cloned successfully into expression vector pcDNA3.1. Sequence analysis indicated the recombinant plasmids were 100% homology with the sequence of FMDV in GenBank. The average diameters of conducted nanopartieles were about 530 nm detected by particle sizer. The stability analysis showed that the nanoparticles were trans- parent, well-distributed and deposit-free after stored at 4℃ for 24 hours. The transfection efficiency of the CaPi-pDNA-GFP on human embryonic kidney 293 （HEK 293） cells in vitro was about 9.6%. The recombinant new gene plasmid embedded multi-shell calcium phosphate nanoparticles was successfully constructed. These results encouraged further research for the development of DNA vaccines against FMDV ＂ O＂ and provided the basis of research for DNA vaccines.