| Newcastle disease(ND)and chicken infectious bronchitis(IB)are the animal infectious diseases that must be notified by the World Organization for Animal Health(OIE)and are ani mal diseases of category I and II respectively as stipulated by the Ministry of Agriculture of C hina.Epidemic diseases can cause serious consequences such as reduced production performa nce and chicken mortality,directly causing huge economic losses,it has become a major threa t to waterfowl farming.However,the most efficient and economical way to prevent and contr ol these two types of diseases is undoubtedly immunization with vaccines;The main types of traditional vaccines include: inactivated and live attenuated vaccines.However,in the face of a virus strain with numerous serotypes like IB,the protection of traditional vaccines is somew hat overwhelming.Moreover,the stability of traditional vaccine strains is poor,and the virule nce often returns to the strong phenomenon.High production costs,complex preparation proc esses and cumbersome immunization procedures have also led to uneven levels of commercia lized vaccines.The amplification of many problems has accelerated the research of new vacci nes,with genetically engineered vaccines taking the lead and gradually becoming the new fav orite in vaccine research.Vaccines that can express multiple epitopes associated with the target antigen and adjuva nt at the same time are known as cocktail vaccines,It is also known as a multiple epitope vacc ine.As the leading researcher in the field of genetically engineered vaccines,which can be sp ecifically recognized and bound by MHC molecules of multiple genetic backgrounds.inducin g an efficient delivery response,enhancing the immune response,and is uniquely positioned t o respond to genetic mutations in pathogenic microorganisms and to overcome adverse factor s in the immune response.In this study,a multi-epitope vaccine rNDV-IBV-T/B strain of infe ctious bronchitis virus expressed by recombinant Newcastle disease virus,which has been con structed by our team,was used.It uses the La Sota strain of NDV as a vector,and first replace s the heat-resistant HN gene of the NDV-TS09-C strain with the La Sota strain to enhance its thermal stability.Then genetic engineering techniques were applied to insert functional epitop es of T and B lymphocytes of IBV-S1 protein between F and M genes of NDV,and the recom binant virus was rescued in vitro by reverse genetic techniques to obtain recombinant virus r N DV-IBV-T/B strain.The recombinant virus rNDV-IBV-T/B strain was effective against Newc astle disease virus and many subtypes of IBV infections.As a genetically modified microorganism,the issue of genetic safety has been a major co ncern in genetically engineered vaccine research.To prevent unnecessary damage to the surro unding environment and people,the Regulations of the Ministry of Agriculture on the Safety Management of Genetically Modified Organisms(GMOs)have also made clear provisions on the production and application of GMOs.As a necessary and important part of the production and application of genetically engineered vaccines,generally,after passing the three stages of small-scale intermediate experiments,medium-scale environmental release experiments,and large-scale production experiments,the GMO safety certificate can be obtained and the next s tage of research can be carried out only after the acceptance.For this reason,this study was co nducted to evaluate the safety of the recombinant virus rNDV-IBV-T/B strain,which provides data support and assurance for the production application of vaccines.At the same time,the d evelopment process of genetically engineered vaccines has been strongly promoted.In this study,the expression and genetic stability of the target gene of the constructed an d preserved recombinant virus rNDV-IBV-T/B strain were analyzed.The recombinant virus was continuously replicated and amplified using the chicken embryo passaging method,and t he chicken embryo allantoic fluid was collected after 25 passages,and the recombinant virus was tested by RT-PCR and sent for sequential analysis of gene mutation.At the same time,sp ecificity detection and sensitivity analysis was performed for the recombinant virus exogenou s insertion gene.The results show that,when the recombinant virus was passed through sever al generations using chicken embryos without mutations and was able to amplify two specific bands of 1592 bp and 977 bp in size.A preliminary indication that recombinant viruses have good genetic stability.Two specific bands were amplified when the c DNA template of the rec ombinant virus was diluted to 10-4,while PCR amplification with other viruses with specific primers did not produce any target bands.The recombinant virus rNDV-IBV-T/B strain was a ble to amplify two target bands and the parent La sota strain was able to obtain a target band.The recombinant virus was also detected with high viral titers using chicken embryos.These r esults indicate that the recombinant virus has a high replication capacity and virulence level e ven after multiple passages,and the inserted genes are stably expressed and specific.One-day-old chicks were vaccinated with nasal drops and eye drops,and the immunizati on dose was 106EID50/0.2m L.Observe the mental status and morbidity and mortality of chic kens daily after immunization.Dissection for observation on days 3,7,14,21,28,35,42,49 and 56 after immunization,and take the chicken tissue organs and environmental drinking wat er,feed,etc.for RT-PCR detection.It was found that there was no chicken mortality during th e whole experiment,good feeding and mental condition of chickens.The chicken tissues and organs were normal on autopsy.RT-PCR results showed that,the target gene of recombinant virus rNDV-IBV-T/B strain was not detected in the organs of the immunized chickens and in the drinking water and feed of the surrounding environment during the whole immunization o bservation period.For further testing to confirm the reliability of PCR results,further evaluati on of the safety of recombinant virus rNDV-IBV-T/B,we used indirect immunofluorescence t echnique(IFA),retention of recombinant viruses in the organism and the environment re-deli neated by cellular level assays.Tissues and organs of target chickens and the surrounding envi ronment were taken on days 3,7,14,21,28,35,42,49 and 56 after immunization and treated with drinking water and feed to infect Hela cells.Recombinant virus rNDV-IBV-T/B and ND V strong virulent Herts33 were also used to infect Hela cells as two positive controls.After te sting we could find that no green fluorescent signal was detected in the tissue organ and surro unding environment samples within 56 d after immunization,and the positive group had brigh t green fluorescent signal markers,i.e.no recombinant virus was detected in the tissue and sur rounding environment,indicating that the recombinant virus was safe for the target animal chi ckens and the environment.In summary,this experiment has objectively evaluated the safety of the epitope vaccine s train rNDV-IBV-T/B,confirmed its safety,and provided data support for promoting the devel opment of vaccines. |
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