Study On Expressing Recombinant Foot-and-mouth Disease Virus Structural Protein VP1 By Transgenic Silkworm Fat Body Bioreactor

Foot and mouth disease is a acute,pyretic and high-tangent infectious disease of artiodactyls,which is caused by foot and mouth virus.It violates artiodactyls violates human and other animals Occasionally. The characteristics of FMD is fast popular, wide spread, acute onset and large harmful.In affected areas, incidence of FMD is come up to 50%-100%,which is harmful to producing of Animal Husbandry.Nowadays inactivated vaccine is the primary foot-and-mouth disease vaccine,but inactivated vaccine is genetic instability, and the effective epitokes may be damage or change in the inactivation processing.The immune effect of nactivated vaccine is short.It may cause toxic or potentially detrimental body's immune response requires multiple injections and needs many antigens and high cost.Inactivated vaccine is comparison of genetic engineering vaccine with higher security,lower cost,large-scale operation.The silkworm is the only ecomomic insect which is domesticated by human.As the newly bioreactor, the silkgland and fat body have such advantages as high efficiency of protein expression and post-modification of translated protein. They can be widely used to produce useful protein of low cost. In this research, we constructed FMDV-VP1 transgenic expression vector which was activated by LP3 promoter and based on piggyBack transposon.Transgenic silkworm was got by micro-injection.In order to supply the useful information for the developing the newly recombinant vaccine which was produced by the transgenic silkworm, we study on the feasibility of using the silkworm's fat body to express the viral antigens of animal.1 Cloning FMDV-VP1 gene and constructing transgenic expression vector.Based on the sequence of the FMDV-VP1 gene, we plused a BamHI restriction enzyme site in the gene 5'end,a NotI restriction enzyme site in the gene 3'end and a 6X histidine tag sequence by primers.A fragment which contains 645bp was got through PCR.Then the fragment was cloned into the PMD18-T simple vector and confirmed by sequencing. After that pMD18-FMDV-VP1 vector was disgested by restrictive enzyme BamHI and NotI and ligated to pSL[LP3-MCS-SV40] vector which was treated by BamHI and NotI for getting pSL[LP3-FMDV-VP1-SV40] vector. Through digesting pSL[LP3-FMDV-VP1-SV40] vector by restrictive enzyme AscI and ligateing the LP3-FMDV-VP1-SV40 fragment into transgenic expression vector pBac[3xp3-EGFPafm] which was treated by restrictive enzyme AscI,and we finally got transgenic expression vector pBac[3xp3-EGFP, LP3-FMDV-VP1-SV40].2 Getting transgenic silkworm of expressing recombinant FMDV-VP1 protein.Ultrapure recombinant plasmid of expression vector was got by using QIAGEN Plasimd Mini Kit. Mixing the helper plasmid and the expression vector by 1:1 and adjusting the concentration to 400ug/μl then injecting the mixture into non-diapaused silkworm eggs to get the transgenic silkworm.34 populations of first generation were detected by the fluorescence microscope during the period of the 5th to 7th of embryo stage. We got 13 positive populations. The rate of positive population is 38%.3 olecular detection of transgenic individualsGenome of G1 generation's silkworm was extracted.The result of inverse PCR and Southern bolt shows that the insert fragments of two transgenic silkworms were located in the 16th and 28th chromosome respectively, and they are single copy. Fat body's RNA of G2 generation's silkworm which from the 4th to 7th day of fifth instar was extracted. The result of RT-PCR demonstrated that FMDV-VP1 gene had transcripted highly in the'fat body during this period and reached the peak before the spin time, which is identical with the expression pattern of the LP3 gene.4 etection expression of recombinant proteinThe 7th day of 5th instar larva's fat body was grinded and solved in lysate. Using the non-transgenenic silkworm strain Dazao as control and antibody of the 6X histidine-tag as the probe, the result of western blot showed that the recombinant protein of FMDV-VP1 expressed successfully.