Preparation And Application Of Polyclonal Antibody To Duck LGP2

China is the world’s largest meat duck producer,with duck meat production accounting f or about 69.2%of the world’s total duck meat production.In recent years,a variety of waterfo wl traditional viruses such as Duck Plague Virus,Duck viral hepatitis virus infected duck floc k morbidity and Duck Tambusu virus and Novel reovirus and other new viruses in the clinical epidemic,to China’s duck breeding industry to bring serious economic losses.Traditional met hods of combating viruses are based on vaccine prevention and antiviral drug therapy,but bot h tend to be specific and unable to achieve broad-spectrum antivirals.In contrast,the innate im mune response of the host,which senses pathogen-associated molecular patterns through patte rn recognition receptors,initiates downstream signaling networks to act as an antiviral,and th e same antiviral pathway has the property of regulating multiple viral replication,offering the possibility of novel broad-spectrum antiviral therapies.Laboratory of genetics and physiology 2（LGP2）is a member of the Retinoic acid induci ble gene I-like receptors（RLRs）family,which,together with two other members of the famil y,RIG-I,and Melanoma differentiation associated gene 5（MDA5）,work together to mediate the antiviral signaling pathway.There are few studies on duck LGP2,and accurate detection o f its expression changes can better reveal whether LGP2 is involved in the host’s antiviral inna te immune response after different viral infections.Because there is no commercial LGP2 anti body for duck,previous studies have mainly detected the nucleic acid level of duck LGP2 by PCR.Therefore,in this study,we hope to establish a method to detect the protein level expres sion changes of duck LGP2 in the host natural immune effect through the preparation of duck LGP2 polyclonal antibodies,and to start the relevant experiments with the clinical material to detect the isolated virus as an example.The experiments consisted of four main aspects as foll owing:Part I:Construction of prokaryotic expression vector for LGP2 in Cherry Valley meat du ck.In this experiment,RNA was extracted from the spleen of healthy cherry duck and revers e transcribed to c DNA as a template for amplification of LGP2 gene,and PCR reaction was pe rformed with amplification primers designed according to the predicted sequence of duck LG P2 on NCBI,and the PCR product was electrophoresed and the target fragment gel was recov ered and sent for sequencing to finally obtain the target gene LGP2.The target fragment was h omologously recombined with the linearized vector by double digestion,and the homologous recombination product was transformed into DH5αreceptor cells,cultured overnight,and then a single smooth colony was picked out for PCR identification and sent for sequencing.The co nstructed recombinant plasmid p ET32a（+）-du LGP2-His was further confirmed to be successfu lly expressed by SDS-PAGE and Western Blot.In order to obtain the recombinant protein wit h high expression,this experiment explored and optimized the conditions for the induction of expression of duck LGP2 recombinant protein,and the results showed that:when the bacteria were cultured at 37°C,220 rpm shaker for about 2 h,the OD_600nm of the bacterial solution was0.4-0.8,then the final concentration of 1.0 mmol/L of the inducer IPTG was added to the bact erial solution,and then placed at 30°C,220 rpm shaker to induce the expression for 6 h.The h ighest expression of the target protein was achieved after 6 h of induction expression in a 220rpm shaker.Part II:Preparation of polyclonal antibodies to LGP2 in Cherry Valley meat duck.In the process of preparing polyclonal antibodies,inclusion body verification of the reco mbinant protein was first performed to determine that the duck LGP2 recombinant protein wa s expressed as an inclusion body protein.In the process of purification of this inclusion body p rotein,the target protein was a fusion protein with His tag,so Ni columns with high affinity fo r His tag were used for the purification of this protein.In order to achieve the desired effect of protein purification,the optimal concentration of imidazole in the elution buffer was firstly in vestigated,and the gradual washing down of the target protein was started when the concentra tion of imidazole in the elution buffer was 500 m M as detected by SDS-PAGE.Protein purific ation was performed with 500 m M concentration of imidazole,and the highest concentration o f protein eluate was found in the second tube after purification.The purified protein was emul sified completely with an equal volume of Freund’s adjuvant after concentration detection,and then inoculated with 4-6 weeks old female BALB/c mice by subcutaneous injection at multipl e points on the back,and blood was collected from the eyes 10 days after the fourth immuniza tion.The potency of the prepared LGP2 polyclonal antibody was detected by indirect ELISA using the primary antibody,and the results showed that the potency was up to 1:64,000,which was high,and its specificity was proved by Western Blot.The duck LGP2 polyclonal antibod ies prepared in this experiment can detect the changes in the protein level of duck LGP2 durin g the antiviral process after the host or cells are infected with virus,which is very important fo r the research on the mechanism of its role in the natural immune system mediated by RLRs.Part III:Detection of clinical disease materialOur laboratory conducted PCR identification of 130 sent disease materials sent from Dez hou,Liaocheng,Weifang,and Heze in Shandong Province from June to December 2020,and tested eight common viruses,Du CV,FAV,DPV,DTMUV,NDRV,H9N2,DHV-Ⅰ,and DHV-Ⅲ,of which 77 were positive,and the total detection rate of viruses was 59.23%.And DTM UV virus was isolated from the positive disease material containing only DTMUV.Among th e detection results,Du CV had the highest detection rate among DNA viruses,followed by FA V and DPV,while among RNA viruses,DTMUV had the highest detection rate,followed by NDRV,H9N2,DHV-Ⅰand DHV-Ⅲ.Part IV:Application of Cherry Valley Meat Duck LGP2 Polyclonal AntibodyIn this experiment,the prepared duck LGP2 polyclonal antibody was used as primary ant ibody for Western Blot validation experiments.Firstly,the interfering RNA of duck LGP2 wa s designed,and the interfering RNA was transfected into DEF cells,and the cells were lysed w ith cell lysis solution at 36 h after transfection,and this was used as the sample for Western Bl ot experiments with LGP2 polyclonal antibody as primary antibody.The results showed that t he expression of LGP2 in DEF cells was significantly lower after interference with RNA than before,which was consistent with the expected results,indicating that the prepared Polyclonal antibodies were effective and specific.Secondly,in this study,DEF cells were infected with DTMUV isolated from clinical material assay,and the cell samples were collected at 12 hours post infection,24 hpi and 36 hpi,respectively,and centrifuged with cell lysis solution and use d as samples for Western Blot experiments.The results showed that the expression of LGP2 w as significantly upregulated at 12 h,24 h and 36 h after infection with DTMUV,indicating tha t LGP2 plays its own role in the host’s natural immunity against DTMUV.In summary,this experiment constructed a prokaryotic expression recombinant plasmid p ET32a（+）-du LGP2-His,further prepared a polyclonal antibody with good specificity for duck LGP2,and established an assay to study the protein level of duck LGP2 in the role of host anti viral natural immunity;verified the practicality of the assay by detecting duck LGP2 knockdo wn cell lines.Tambusu virus was isolated by testing clinical material,and DEF cells were infe cted with the isolated virus,and the involvement of LGP2 in host innate immunity against tam busu virus infection was verified by the assay.This experiment provides experimental materia ls for the in-depth study of the interaction between LGP2 and virus and the development of no vel antiviral drugs or vaccine adjuvants based on this,which has more important clinical signi ficance for the prevention and treatment of infectious diseases in duck.