Recombinat Expression Protein VP60in E.coil, Preparation Of Its Polyclonal Antbody And Transformation Of VP60Gene In Plants

Rabbit hemorrhagic disease (RHD) is caused by the Rabbit Hemorrhagic Disease (RHDV). It is a highly contagious and acute disease with high morbidity and mortality in adult rabbits. The disease is characterized by severe liver damage in combination with a disseminated intravascular coagulation syndrome leading to hemorrhaging in different organs of adult rabbit. The RHDV genome is a single-stranded, positive-sense RNA molecule,7,437nt in length. Sequencing of the genome has revealed the presence of two open reading frames (ORFs). ORF1encodes a polyprotein of257kDa and includes the capsid protein, VP60, at its C-terminus. ORF2is located at the3'end of the genome and encodes a small capsid protein, VP10. The VP60capsid protein, whose molecular size is about60kDa, has various functions, including assembly into the capsid, as well as interaction with host proteins to mediate host-cell receptor binding and antigenic diversity, conferring host specificity and antigenicity. At present, there are several available vaccines against rabbit hemorrhagic disease on the market, which is the main component of commercially used vaccines isolated from liver extracts of infected rabbit or recombinant protein VP60expressed in several heterologous systems like Escherichia coli, Saccaromyces cerevisiae and so on. Although they have proved to be effective tools for prevention of the disease, there are many problems resulting from the use of infectious virus and the risk of its dissemination from vaccine factories. Thus, it is important to develop alternative approaches for producing vaccines. In recent years, transgenic plants have demonstrated considerable potential for the production of vaccines. Plants are a promising platform for the production of vaccines.This investigation, based on the clone of vp60gene of RHDV, the expression of recombinant protein VP60in Escherichia coli and preparation of its polyclonal antibody were studied, tobacco and Lotus corniculatus L. were transformed by Agrobacterium tumefaciens with the gene vp60, and also the regeneration of alfalfa (Medicago sativa. Golden Empress) was explored. The major results are as follows:1. The rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) gene was obtained by PCR, and cloned into prokaryotic expression vector pET28a(+). The identified construct was transformed into E. coli and over-expressed protein was purified by nickel chelating chromatography and polyclonal antiserum was raised against rabbit. The vp60gene was amplified by PCR. The recombinant prokaryotic expression vector pET28a(+)-vp60was constructed by molecular technique, and then the recombinant was transformed into E. coli BL21(DE3) to induce protein expression with IPTG. The recombinant protein purified by nickel chelating chromatography was used as antigen to immunize rabbit for preparation of polyclonal antibody. The protein VP60polyclonal antibody has provided reliable tools for future study in the transgenic plant of vp60.2. The VP60gene from RHDV (rabbit hemorrhagic disease virus) YL Strain in Northeast of China, under control of the ast1A promoter from Rubisco small subunite genes of Arabidopsis thaliana, was introduced into the T-DNA region of plant transfer vector pCAMBIA1300and transferred to tobacco(Nicotiana tabacum cv. Petit Havanna SR1)with Agrobacterium tumefaciens-mediated method. PCR and RT-PCR analysis of the transformed tobacco plants confirmed the integration of the vp60gene copy into the plant DNA and vp60gene transcription produced. Western blot analysis revealed that the VP60protein was expressed in tobacco under control of ast1A promoter.3. The regeneration system of Lotus corniculatus L. was established. On the medium MS+6-BA0.2mg/L, the hypocotyls and the cotyledon from Lotus corniculatus L. were induced easily and directly to be buds with regeneration rate100%and93.3%respectively. The regeneration plants were rooted on the medium MS0or MS0+NAA0.1mg/L. The vp60gene from RHDV (rabbit hemorrhagic disease virus) YL Strain in Northeast of China was introduced into the T-DNA region of plant transfer vector pCAMBIA1301transferred to hypocotyls of Lotus corniculatus L. Agrobacterium tumefaciens-mediated method. PCR and RT-PCR analysis of the transformed Lotus corniculatus L. plants which grew on the medium MS0+6-BA0.2mg/L+hyg2mg/L, confirmed the integration of the vp60gene copy into the plant DNA and vp60gene transcription produced.4. The alfalfa (Golden Empress) was studied on establishing the regeneration system. The best time for the disinfection was lmin in75%alcohol, then5min in0.1%mercuric chloride. The experiment studied the effects of different hormones variety and ratio for the alfalfa on the callus induction and induced differentiation. The optimal medium for callus differentiation was MS+NAA1.8mg/L+6-BA1.6mg/L, the optimal medium for bud differentiation was MS+6-BA0.6mg/L+NAA0.3mg/L. Induced differentiation of hypocotyls of seedlings were put on the mediums including different concentration of hygromycin for optimal screening. Different concentrations of hygromycin had some restraining effects on callus inducement and differentiation in alfalfa hypocotyl, and the restraining effect enhanced along with the rising concentration of hygromycin, and the concentration for selecting was15mg/L.