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Foot-and-mouth Disease Virus (FMDV) VP1 Gene Recombined and Expressed in Model Plant Chloroplasts

Author: SunMeng
Tutor: QianKaiXian；ShenGuiFang
School: Zhejiang University
Course: Cell Biology
Keywords: foot-and-mouth disease virus (FMDV) VP1 protein tobacco cholera toxin Chlamydomonas reinhardtii chloroplast transforma
CLC: Q785
Type: PhD thesis
Year: 2003
Downloads: 225
Quote: 5
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Abstract

Foot-and-mouth disease virus (FMDV) is the etiological agent of an important disease of livestock. Vaccination is the major method to prevent FMD. Currently, FMD vaccines are either conventionally inactivated vaccines or attenuated vaccines. Both types of vaccines are not completely safe and pose some fatality risks. As a result, developing a safe and effective FMDV vaccine has become a research priority.The structural protein VP1 of FMDV carries critical epitopes which can induce neutralizing antibodies. The use of transgenic plants for producing FMDV VP1 antigen protein has been reported and may prove to be safer than conventional vaccines. Recently, FMDV VP1 protein had be expressed in transgenic plants. Moreover, mice feeding on FMDV-VP1 transgenic plant leaves in their diet produced a virus-specific immune response that could protect the immunized mice against FMDV challenge. Transgenic plants, as a vaccine source, have several advantages in which they are relatively inexpensive, safe and can be adminstrated orally. However, in the reports the expression of VP1 antigen in the transgenic plants was very low (0.01%~0.37% total soluble protein (TSP) ), thus limiting the further application of a plant expression system.Recently, the use of plant chloroplasts to express valuable proteins has been suggested and has several practical advantages compared to that of nuclear expression systems. These include the ability to target the gene of interest to site-specific areas of the chloroplast genome, high-level expression of foreign genes and increased environment security . In this study, we transformed the VP1 gene of FMDV serotype O into the tobacco chloroplast and Chlamydomonas reinhardtii chloroplast, and achieved highly expression levels, paving the way for engineering an FMDV vaccine via transplastomic plants.In the second part of the thesis, we described that a tobacco chloroplast expression vector, PTRVP1, containing the foot and mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene was constructed and transfered to the tobacci chloroplast genome by the biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. To determine the site-specific integration of the foreign gene into the chloroplast genome and the level of homogeneity, the three transformation lines of the second selective generation were analysized through PCR (PCR-Southern blot)analysis using the primers of FMDV VP1 gene and homologous fragment of tobacco chloroplast, respectively. The results of PCR (PCR-Southern blot) analysis showed that the foreign genes had integrated into the chloroplast genome of the three transformants through the homologous recombination. Western blot and ELISA assays indicated that FMDV VP1 gene was expressed in tobacco chloroplasts and accounted for 2%~3% of the total soluble protein (about 20-30 (ig of chloroplast-expressed VP1 protein in 1 mg of total soluble protein).In the third part of the thesis, A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVPl, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed. Transformation of C. reinhardtii chloroplast was achieved by biolistic bombardment with pACTBVPl. 10 clones resistant to spectinomycin were obtained, 3 resistant transformants were subjected to three rounds of Spc selection. To determine that the integration of foreign genes had occurred hi the chloroplast genome at the directed site by homologous recombiantion and to evaluate the degree of homogeneity of the C. reinhardtii transformants, PCR and PCR-Southern blot analysis were performed using the primers of C. reinhardtii chloroplast homologous fragment. The result demonstrated that the CTBVPl fusion gene was inserted into the C. reinhardtii chloroplast genome and after three rounds of Spc selection the recombinant chloroplast DNAs were the majority, and the wild-type chlorop

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