Study On Large-scale Cultivation Of IBDV In Bioreactor System And Production Of Effective Attenuated Vaccine

Infectious bursal disease (IBD) which caused by infectious bursal disease virus (IBDV) is anactue and highly contagious immunosuppressive disease in young chickens. It was still one of themost serious avian diseases, since nineteen eighties entered into China.At present,It usuallyprevented and controlled by means of vaccination. In order to protect chickling from infection ofIBDV during the brooding period, the breeders are usually injected with inactivated vaccines tomake chickling acquire high maternal antibody. However, the immunological effect of the vaccineis ineffective due to the frequent variation of IBDV and the low titre of vaccine virus. At present,domestic henneries mostly imported inactivated vaccine. In recent years, biological reactorlarge-scale cultivation technique had used for veterinary biological products. But little studied onthe IBDV, It reported also domestic research using Vero cells in biological reactor forproliferation IBDV, but, it couldn’t be applied.According to the need of study on high efficientvaccine for IBD,through suspension culture by the carrier in bioreactor and DF-1cell lines,compared with the traditional culture method, viral titer of IBDV improved100times and inorder to meet the needs of the market, it successful developed a kind of high quality and efficientIBD inactivated vaccine.1. According to the need of detection on viral titer of IBDV under high efficient culture,A SYBRGreen I real-time RT-PCR (QRT-PCR) was successfully developed using a pair of primersspecific to the conserved region of VP4gene of IBDV and compared with TCID50method bymonitoring the proliferation dynamic of IBDV in DF-1cell line adherent to micro carrier intubular reactor. And by the QRT-PCR assay and TCID50detection there was a parallel correlationof IBDV proliferation dynamic in DF-1cell cultured in cell bottles. However, the QRT-PCR assaywas more rapid and sensitive than the TCID50method.2. This study comprehensively researched on the proliferation characters of IBDV on DF-1cell atmicro carrier in tubular miniature reactor and AP10bioreactors by established QRT-PCR assayand compared with the classic TCID50determination method.conclusion:①Inoculated with3x107or so in tubular(built-in0.6g Paper carrier), DF-1cells increased to2.4~2.5x108after6days, andwhich was determined as the best inoculation time for IBDV; optimal inoculation dose of IBDVwas0.79MOI, the best time of collection was at28h after virus inoculation; There was a parallelcorrelation of virus titers between supernatant and cell suspension, but the latter was morehigh,and could achieve higher than109.5TCID50/ml.②Increment of DF-1cells and sugerconsumption had obvious parallel correlation, the average sugar consumption of each cell in cell growth period (144h) was up to6.5x10-10g/24h, according to the sugar consumption rate wecould calculated the number and estimated growth state of DF-1cells;the peak time of sugarconsumption prior to virus titers’ after inoculation,when the sugar consumption fall from pesks tozero instantaneity, it could reach the higher virus titers.③It could again obtaint a peak9.5(-lgTCID50/ml) at9h after collecting1/2of viral solution at peak time, and harvesting1/3at18h.④In the bioreactor the titer of IBDV achieved9.5(-lgTCID50/mL) at24h after inoculation bythe doses of1.05MOI; and at8h after taken out1/2supernatant its titeronly reached9.25(-lgTCID50/mL).⑤In the reactor, the peak of suger consumption was earlies than the titers’,whenthe consumption of sugar content decreased0,titers of IBDV reached the peaks, and it wasconcerned with inoculation cell numbers and doses of IBDV.⑥The rise and fall of the peak timeof IBDV under the high density culture basically had agreement byTCID50and QRT-PCR assay,but the QRT-PCR assay was more rapid and sensitive than the TCID50method.3. IBDV HQ strains continuously passaged culture on DF-1cell lines of the chick embryo until10generations, and viral content, immunogenicity, determination of specificity test preservationperiod and pure inspection were determined for cell virus of IBDVwithin the10generations, andthe cytotoxic properties mentioned above basically had consistency for the10generation, sosuccessfully established virus seed bank for IBDV HQ strain. Using viral solution of IBDV HQstrain cultured in reactor (109.5TCID50/mL)、spining the bottle virus of DF-1cells (108.5TCID50/mL,3times the enrichment) and spining the bottle virus of CEF cells (107.5TCID50/mL,5times theenrichment)producted three kinds of inactivated vaccines according to trial implementation rules,and carried out the inspection. The results show that, testing of the semi-finished and finishedproduct (physical properties test, sterility test, safety test) completely conformed Coderequirements. After the three kinds of vaccine immunization, neutralizing antibody of DF-1cellsbioreactor vaccines was significantly higher than that of spining bottle ones, protection rate of twoimmune attack group was100℅,and control group was0℅,but neutralizing antibody and attackprotection of CEF cell vaccines relatively belowed them.This thesis researched condition and method under high under density cultivation of DF-1cell at micro carrier in tubular reactor, and exploration optimal culture conditions and toxic way ofIBDV in bioreactor under the automatic control, so this provided a theoretical basis and practicalexperience for industrial large-scale cultivation of IBDV and production of inactivated vaccine, soit is very practical.