Construction, Identification and Expression of Transgenic Alfalfa ...

Abstract

ObjectiveTo make E.granulosus BALB/c model and amplify the Eg95 and EgA31 coding gene for constructing the Eg95-EgA31 fusion gene; To construct recombinant plasmid pBI-Eg95-EgA31; To transform the Agrobacterium tumefaciens(At) with plasmid pBI-Eg95-EgA31; To construct and identify the transgenic alfalfa vaccine of Echinococcus granulosus Eg95-EgA31 fusion gene; To analyse the expression of recombinant plamid pBI-Eg95-EgA31 in alfalfa; To provide the basis for further research of the immune mechanism of the transgenic alfalfa vaccine of Echinococcus granulosus.MethodsE.granulosus BALB/c model had been made by injecting intraperitoneally protoscoleces of Eg. Total RNA was extracted from hydatid cyst protoscoleces shattered by supersound with a nucleic acid extraction kit. Total RNA as template, the primers containing cutting sites of restriction-endonucleases were designed according to the cDNA sequence of Eg95 and EgA31 genes downloaded from GenBank, the Eg95 and the EgA31 coding gene were amplified by RT-PCR, and were identified by sequencing and electrophoresis. By Gene SOEing technique, Eg95-EgA31 fusion gene was constructed by splicing Eg95 gene and EgA31 gene with (Gly4Ser)3, then cloned into plant express vector pBI121 to construct recombinant plasmid pBI-Eg95-EgA31. The recombinant plasmid was introduced into Agrobacterium tumefaciens(At)LBA4404 strain by electroporation. The recombinant Agrobacterium tumefaciens(rAt) was identified by PCR amplification, sequencing and electrophoresis. The alfalfa leaves were infected with the recombinant Agrobacterium tumefaciens(rAt). The alfalfa somatic embryo was picked up from the callus with kanamycin resistance. After the resistant somatic embryo grown into transgenic alfalfa, the Eg95-EgA31 fusion gene expression products were extracted from the leaves. The transgenic alfalfa were confirmed by SDS-PAGE, Western blot, PCR and RT-PCR assay. ResultsEg95 and EgA31 antigen genes amplified were almost same as the corresponding cDNA ORF sequences in GenBank by sequence analyzing. Eg95-EgA31 fusion gene identified by Agarose gel electrophoresis was about 1016bp, which was in accordance with the expected result by sequence analyzing. Restriction-endonuclease digestion demonstrated that the Eg95-EgA31 fusion gene was correctly cloned into the multiple cloning site of plant expression vector pBI121 to construct recombinant plasmid pBI-Eg95-EgA31. It was confirmed that the recombinant plasmid was successfully introduced into Agrobacterium tumefaciens(At) by analyzing the recombinant At(rAt) selected in YEB medium with 10μg/μl kanamycin by PCR, DNA sequencing and electrophoresis.SDS-PAGE and Western blot showed the recombinant plasmid was able to express target protein in alfalfa, which was expressed as a band of 37.5kDa. The content of Eg95-EgA31 contained 0.05% of total leave protein of transgenic alfalfa and could combine with specific antibody in mice serum. The 1016bp fusion gene of the transgenic alfalfa was amplified successfully by PCR and RT-PCR.Conclusions1. Eg95 and EgA31 antigen coding genes were successfully amplified by RT-PCR respectively.2. The Eg95-EgA31 fusion gene was successfully amplified by Gene SOEing technique.3. The recombinant plasmid pBI-Eg95-EgA31 was successfully constructed.4. The Agrobacterium tumefaciens was successfully transformed with the recombinant plasmid pBI-Eg95-EgA31.5. The transgenic alfalfa of Eg95-EgA31 fusion gene was cultivated successfully.6. The recombinant plasmid (pBI-Eg95-EgA31) was able to express target protein in alfalfa, which was of specific antigenicity.