Development Of Monoclonal Antibodies Against Four Plant Viruses And A Vaccine To Infectious Bursal Disease Virus And Their Applications

Expression of infectious bursal disease virus VP2 protein in rice seeds and its immunogenicity and immune protectionInfectious bursal disease virus(IBDV)can cause acute infectious disease,a highly contagious and mortal disease in young chickens with immunosuppression and large scale of loss due to higher susceptibility to other pathogens and immunization defeat.Classic inactive vaccine and attenuated live vaccine have been confronted with the threat of the emergence of virulent IBDV variant since 1980's.Moreover,these classic vaccines have a number of disadvantages.The inactivated vaccine is costly,poorly effective and difficult to administer,while the live attenuated vaccine may result in the emergence of IBDV variants due to virus recombination.Thus,there is an urgent need to develop new cheap and safe vaccines with higher efficacy and fewer side-effects.For elucidate whether rice is suitable as a bioreactor for expression of IBDV VP2 protein and the possibility for using expressed protein as antigen for subunit vaccine against IBDV, infectious bursal disease virus(IBDV)host-protective immunogen VP2 protein was expressed in rice seeds,its immunogenicity and protective capability in chickens were investigated.The 35S promoter in pCambia1301-35S was substituted with the 1920 bp Gt1 promoter of the rice glutelin GluA-2 gene amplified from rice cultivar Xiushui 11 to produce an endosperm-specific expression vector,pCambia1301-Gt1.The VP2 cDNA of IBDV strain ZJ2000 was then cloned into pCambia1301-Gt1 under the control of the Gt1 promoter to produce pCambia1301-Gt1-VP2.Agrobacterium turnefaciens containing the recombinant vector was used to transform rice embryogenic calli.PCR and Southern blot analyses showed that IBDV VP2 gene had been integrated into the genomic DNA of 121 transgenic rice lines and the transformation efficiency was 20%.All the 121 transgenic lines were grown to maturity in a greenhouse.ELISA assay indicated that the expression level of VP2 protein in transgenic rice seeds varied from 0.678%to 4.521%μg/mg of the total soluble seed protein.Western-blot analysis revealed that a band with a molecular mass of about 50 kDa reacted with anti IBDV antiserum in extractions of transgenic rice seeds,but the color of the bands were different in individual lines.The results indicated that transgenic lines expressed different amounts of VP2 protein.The vaccinated chickens were challenged with virulent IBDV BC6/85 21 days post the boost. Chickens were killed 7 days post challenge and anatomised.The results indicated that the chickens vaccinated with high dose(fed 5 g amount of transgenic rice seeds)had a protection rate of 83.33%(5/6).When the doses were decreased to 3 g or 1 g,the protection rate declined to 33.33%(2/6)and 16.67%(1/6),respectively.Virus neutralization titres analysis indicated that transgenic rice seeds expressing VP2 protein could induce high titer neutralizing antibodies against IBDV in chickens vaccinated with high dose(5 g).When the doses were decreased,the neutralizing antibodies titers also declined and a significant dose-effect was observed.Those results show that transgenic rice-derived VP2 protein could induce a strong immunological response and a high level of neutraling antibody in vaccinated chickens.Transgenic rice-derived VP2 protein has good immunogenicity and immune protection as a vaccine.A 5-fold of the highest dose(25 g)was orally administered to six two-week-old chickens each day for a month. No mortality,side effects or signs of any disease were observed during 2 months inspection.The average body weights(3.12 kg)of the chickens fed 25 g of transgenic seeds were not significantly different in body weight from those(3.20 kg)of the control group.These results indicate that transgenic rice seeds expressing IBDV VP2 protein can be used as an effective,safe and inexpensive vaccine against IBDV.Development of monoclonal antibodies against four plant viruses and their applicationsTomato spotted wilt virus(TSWV)is the type member of the genus Tospovirus and causes significant losses in a wide range of economically important ornamental and vegetable crops worldwide.TSWV is transmitted by thrips to over 1000 susceptible species of monocotyledonous and dicotyledonous plants in temperate and tropical regions.The about 29-kDa nucleocapsid protein gene(777 bp),located on the ambisense S RNA segment of TSWV OiGpTs1 isolate,was cloned with RT-PCR with a pair of TSWV-specific primers.The cloned gene was expressed using pET-32a expression system in Escherichia coli and the expression of recombinant protein was verified by Western blot using the anti-TSWV antibody.The recombinan protein was purified using Ni-NTA agarose,a metal chelate affinity chromatography and then used for the production of monoclonal antibodies.A panel of three murine monoclonal antibodies(MAbs)against the recombinant nucleocapsid protein of TSWV were produced by hybridoma technology when selected by indirect enzyme-linked immunosorbent assay(ELISA).Western blot assays indicated that three MAbs reacted specifically with the virus nucleocapsid protein band at molecular weight of approximately 29 kDa.Triple antibody sandwich(TAS)ELISA and immunocapture RT-PCR (IC-RT-PCR)methods were then established for reliable and efficient detection of the TSWV isolates using the produced MAbs.When 250 field samples collected from Zhejiang and Yunnan provinces were tested by TAS-ELISA,no samples were found to be infected by TSWV with the exception for the positive controls.In IC-RT-PCR using the MAbs and specific primers in the region of the nucleocapsid protein gene,positive samples gave one specific band about 777 bp in length.The validity and reliability of the results of IC-RT-PCR was confirmed by sequencing analysis.Five hybridoma cell lines secretingMAbs against Carnation mottle virus(CarMV)were produced by fusing mouse myeloma cells(SP2/0)with spleen cells from BALB/c mice immunized by the CarMV particles.The five MAbs could specifically react with CarMV.The titres of ascitic fluids of the five MAbs are 10^-6in indirect antigen-coated plate(ACP-)ELISA. Isotypes and subclasses of 3G1,1B9,2A9 and 2F8 belong to IgG1 while that of 2F2 belong to IgG3.Western-blot analysis showed that all the five MAbs can react specifically with the 38 kDa coat protein submit of CarMV.The 2A9 was used in ACP-ELISA for detection of CarMV. ACP-ELISA could successfully detect 0.1 ng purified CarMV or virus in plant sap diluted 1:800. The presence of CarMV in field Dianthus caryophyllus tissues was investigated with ACP-ELISA and the detections showed CarMV are common in field samples of Dianthus caryophyllus.Two hybridoma cell lines,2B12 and 4E7,secreting MAbs against Potato virus X(PVX) were produced by fusing mouse myeloma cells(SP2/0)with spleen cells from BALB/c immunized by the PVX particles.The two MAbs could react specifically with PVX and the titres of their ascitic fluids were up to 10^-6in ELISA.Isotypes and subclasses of both 2B12 and 4E7 were found to be allied to IgG1.The two MAbs were used in ACP-ELISA for PVX detection and ACP-ELISA could successfully detect the amount of purified PVX as low as 0.2 ng or virus in plant sap diluted to a ratio of 1:600.The presence of PVX in field potato tissues was investigated with ACP-ELISA and the detection results showed that PVX is common in field samples of potatoes.Four hybridoma cell lines,2A2,5149,5H2 and 5E12,secreting MAbs against Lily symptomless virus(LSV)were produced by fusing mouse myeloma cells(SP2/0)with spleen cells from BALB/C immunized by the LSV particles.The four MAbs could specifically react with LSV,Western-blot analysis showed that all the four MAbs(2A2,5H9,5H2 and 5E12)to LSV can react specifically with the 32 kDa coat protein of LSV.The titres of ascitic fluids of the four MAbs are up to 10^-6in ELISA.Isotypes and subclasses of 5H9 and 5E12 belong to IgG1 while those of 2A2 and 5H2 belong to IgG3.Isotypes of light strains of the four MAbs all belong toκ. The four MAbs were used in ACP-ELISA for LSV detection,and ACP-ELISA could successfully detect 1.8 ng of purified LSV or virus in plant sap diluted 1:300.The presence of LSV in field lily tissues was investigated with ACP-ELISA and LSV are common in field samples of lily.