Monoclonal Antibodies Of Chicken SP-A And Histopathological Changes In Avian Viral Infections

Chicken pulmonary surfactant protein A(cSP-A)is a hydrophilic glycolprotein,which is secreted by the alveolar pneumocytes and belongs to C-type lectin family.cSP-A plays an important role in the pulmonary innate immune system and has some homology with mammals SP-A.However,the distribution of cSP-A in tissue cells and its antipathogen immunity mechanisms are not clear.The main objective of this study was to express the soluble cSP-A protein by prokaryotic systems for the preparation of its monoclonal antibody,and to study the distribution of cSP-A in tissue cells for the further anti-viral mechanism and application in clinical medicine.The specific contents and results are as follows: 1.Soluble expression and detection of cSP-AIn the study,cSP-A gene was amplified by RT-PCR and then subcloned into p MD19-T simple vector.After sequencing,the mature cSP-A fragment without the signal peptide sequence was transferred into the prokaryotic expression vector,p Cold-TF.The final constructor is designated as p Cold-cSP-A.The plasmid was transformed into E.coli BL21(DE3)competent cells.Induced with Isopropyl ?-D-1-thiogalactopyranoside(IPTG),the bacteria were grown in LB broth at 16°C for 24 hours.The products were sonicated and then purified by His-bind purification kit.The results showed that the fused protein were soluble expressed with a size of 80 KDa by SDS-PAGE analysis and by Western Blot assay.However,the fused peptide had no activities to inhibit the tested bacteria.2.Preparation and identification of monoclonal antibodies to cSP-AIn this study,to prepare the monoclonal antibodies against cSP-A,we cloned the p Cold-cSP-A and immunized the mice with the production of the prokaryotic expression.The eukaryotic expression vector pc DNA3.1-cSP-A were transfected into 293 T cells to screening antibodies against cSP-A by indirect immunofluorescence assay(IFA).At last,3 specific monoclonal antibodies were obtained,named as 1D12,2F1 and 5C8.Screening by IFA,indirect ELISA and Western Blot analysis,1D12 had the highest antibody titer and the most satisfactory immunoreactivity.The preparation of these antibodies established the foundation of the further research of the biological functions of the cSP-A.The sub-types of 2F1 and 5C8 were detect to be Ig G2 a,1D12 was Ig G1.The light chain was ?.Results showed that the antigenic epitope of 1D12 and 5C8 were 45-60 aa.3.Establishment and detection of cSP-A using SYBR Green quantitative RT-PCR assayBased on the cSP-A gene sequences in Gen Bank,a pair of 153 bp specific primer was designed and the reference gene was c?-actin(Accession No.is JN639846).The recombinant plasmid pc DNA3.1-cSP-A was used as positive standard to establish a SYBR Green RT-PCR to analyze the expression characteristics in lung,treachea,bronchi,laryngopharynx,air sac,liver,kidney,spleen,pancreas,duodenum,cecum and bursa of SPF chicken.Regarding the Ct value of bursa as the control and using 2(-??Ct)relative quantitative method to analyze the relative expression of cSP-A gene in various tissues.Results showed that the melting curve was one specific peak,and the standard curve was Y=-3.5832X+36.07(R2=0.99303).The RT-PCR was highly sensitive to 1.58×102 copies/?L.The q RT-PCR revealed that the expression level of cSP-A gene in the respiratory system was lung>trachea> bronchi >laryngopharynxa> air saca> kidney.And cSP-A had low levels of expression in the liver,spleen and pancreas.4.Co-infection of avian respiratory viruses decreased SP-A expression level in chickens4-week-old SPF chickens were inoculated with strain M41 of Infectious Bronchitis virus(IBV),strain C1 of H9N2 subtype avian influenza virus,strain F48E9 of Newcastle disease virus(NDV)or IBV plus H9N2 virus,respectively.Control SPF chickens were inoculated by 0.01 M PBS instead of virus infections.Tissue samples(lung,treachea,bronchi,laryngopharynx,air sac,liver,kidney,spleen,pancreas,duodenum,cecum and bursa)were collected at 72 hours post-infection,which were separated into two parts: either immediately frozen in liquid nitrogen or placed in 10% neutral formalin.The fixed tissue samples were stained with hematoxylin and eosin reagents.Results of H&E staining showed that lung,treachea,bronchie,laryngopharynx and air sac of virus-inoculated SPF chickens were revealed to have significant lesions by histopathological changes,especially in NDV group.The pathologic changes of H9N2-IBV co-infection group were more sereve than the H9N2 group and IBV group.To examine the cSP-A expression level in chickens,RT-PCR analysis and immunohistochemistry(IHC)assay were performed.cSP-A m RNA and protein expression level were significantly decreased in virus-infected groups compared to the control group,especially in H9N2-IBV co-infection group.The results provide evidence that infection of avian respiratory viruses can decrease cSP-A expression level in chickens,so the expression level of cSP-A is suggested as an indicator in pathological injury.In conclusion,we successfully expressed the soluble cSP-A protein by prokaryotic systems and prepare three clones of monoclonal antibodies.Detected by m Ab 1D12,the distribution of cSP-A in tissues and histopathogenic changes in virus infections were analyzed and identified.These results are helpful for the further study in anti-viral mechanism of cSP-A and its clinical application.