Preparation Of Monoclonal Antibodies Against VP1 Protein Of Chinese Sacbrood Virus And Identification Of Immune Related Genes In Chinese Honeybee,Apis Cerana Cerana(Hymenoptera:Apidae)

Sacbrood Disease?SBD?is one of the most important viral diseases that threaten Apis cerana cerana larvae and lead to serious larvae death and significant colony decline.Chinese Sacbrood Virus?CSBV?is the causative agent of cystic larvae in A.c.cerana.In recent years,it has become one of the most widely distributed viral diseases.In this research,CSBV were investigated thoroughly,and the molecular biological property of CSBV structural protein VP1 is mainly studied.In response to CSBV infection,two immune-related genes,AccSRP9 and AccGABARAP were analyzed by molecular cloning,biological information analysis and prokaryotic expression.The main results are as follows:1.Investigation on the detection and prevalence of CSBV.Using RT-PCR method,common bee viruses have been detected.The highest detection rate of CSBV was 94.59%,followed by BQCV?77.03%?.The detection rate of CSBV showed that CSBV is the highest incidence and threatens Apis cerana cerana.The CSBV were found in different geographical areas and were detected in the northeast,north,central,south,southwest,and eastern parts of the country with varied infection frequencies.The occurrence was consistent with the main honey flow season.Meanwhile,the viruses exhibit a wide range of mixed infections.CSBV is most susceptible to infect larvae during the spring breeding period,showing a trend of increasing gradually from January to April,decreasing from June to September and rising again after October and November.2.Purification and scanning electron microscopy of the CSBV.In combination with the ultra-high speed centrifugation and sucrose density gradient centrifugation and purification method,CSBV was detected from Beijing and Shanxi strains.Using scanning electronic microscope,CSBV-BJ and CSBV-SX virus particles were clearly observed.The virus particles are nearly round shape?diameter of about 30 nm?and spherical icosahedron.3.Preparation of CSBV-VP1 monoclonal antibody.The CSBV-BJ-VP1 gene was cloned and the effective sequence was 945 bp,encoding 315 amino acids,Nbp/3.The predicted molecular weight and isoelectric point of the gene were approximately 35.59 kDa and 9.38,respectively.By prokaryotic expression,the optimal expression conditions(cultured at 37°C,induction with an IPTG concentration of 1.0 mM when the OD₆₀₀ value was 1.0)and a large amount of the recombinant protein pET-32a-BJMY-CSBV-VP1 were obtained.The recombinant protein was immunized to mice,and two strains of VP1 monoclonal antibody ascites were successfully prepared.The subclasses of monoclonal antibodies were IgG2a and the light chain was?chain.4.Cloning,biological information analysis and prokaryotic expression of SRP9 gene from A.c.cerana.Using RT-PCR,the amplified SRP9 gene has a coding region of 237 bp,encoding 78 amino acids?Nbp-3?/3 with a relative molecular mass of 9.36 kDa and an isoelectric point of 9.17.Phylogenetic tree analysis showed that AccSRP9 was clustered with SRP9 of Apis mellifera and Apis florea.Protein secondary structure predictions revealed that it contained 2 alpha-helical regions and 3beta-sheet regions.Homologous modeling yields the three-dimensional structure of the protein.After prokaryotic expression,it was found that the recombinant protein was expressed in inclusion bodies and purified by a method of washing inclusion bodies to obtain a recombinant protein with a GST tag.The expression characteristics of SRP9 gene were studied by qPCR.The result showed that the expression level of the SRP9 gene in the CSBV-inoculated larvae is significantly increased.5.Cloning,biological information analysis and prokaryotic expression of GABARAP gene from A.c.cerana.RT-PCR method was used to amplify the effective sequence of 354 bp,encoding 117 amino acids?Nbp-3?/3 with a predicted molecular mass of 13.99 kDa and an isoelectric point of 9.48.Phylogenetic tree analysis showed that GABARAP is in the same cluster as other insects of the same order,such as A.mellifera,A.florea,Apis dorsata,Bombus terrestris,and Bombus impatiens.Protein secondary structure predictions revealed that it contained 3 alpha helices and 4 beta sheet structures.Homologous modeling yields the three-dimensional structure of the protein.After prokaryotic expression,it was found that the recombinant protein was expressed in inclusion bodies.His-tagged protein purification kit was used to obtain a His-tagged recombinant protein.The expression characteristics of GABARAP gene were studied by qPCR.The result showed that the expression level of the GABARAP gene in the CSBV-inoculated bee larvae is significantly increased.