Establishment Of The Vero-CD150 Cell Line For The Isolation Of Canine Distemper Virus And Preparation Of Monoclonal Antibodies Against The Heamagglutinin And Fusion Protein Of The Virus

Canine distemper (CD) caused by canine distemper virus (CDV) is an acute infectious disease of canine, which is characterized by high pathogenicity and infectivity. CDV belongs to Morbillivirus of Paramyxoviridae. It can infect dog, mustela, coon, and even rare animals such as ailuropoda and ailurus. Recent years, CDV's range of host becomes wider. It is imminent to prevent CD for its high mortality to canine, fur animal and wildlife, etc.1. Construction of the Vero cell line expressing canine CD150 gene and its use in isolation of CDVIsolation and culture of CDV is the fundamental way to make a definite diagnosis of CD. However, CDV's resistance to the environment is weak so that the isolating rate is low. Many agents can effect CDV's isolation such as the position and time of collecting smples, the way of handling samples and the level of ill animals'antibodies. Among the agents the extremely important one is the saitivity and utility of cells which is chosen to isolate the virus. Therefore choosing a kind of suitable cell to cultivate CDV is the key point. Vero cell line is the preferred one to isolate CDV. It needs to passage many times and add pancreatic enzyme in order to get CPE. In fact many virus strains still can't appear CPE , which makes it inconvenient to isolate CDV. It is reported that signaling lymphocytic activation molecule (SLAM, CD150) is recognized as CDV's receptor, CDV gets into the cell to replicate by the way of recognizing CD150. This discovery will help us to better study the mechanism of the measles virus infection and the different tropism to cells among virus strains'. In this study CD150 cDNA from canine was amplified by RT-PCR and cloned into a eukaryotic expression vector pIRES-neo. The positive plasmid contained CD150 gene was determinded by restriction enzyme analysis and named pIRES-CD150, then transfected into Vero cell line. The stable transfectants were screened by G418 and subcloned by gradient dilution. The Vero-CD150 cell line subclones highly expressing CD150 gene were identified by RT-PCR and flow cytometry (A high antigenicity fragement of CD150 gene was amplified by PCR and was cloned into a prokaryotic expression vector pGEX-6P-1. A fusion protein which was about 50 kDa was constructed and used to prepare polyclonal antibodies for use of flow cytometry by the way of immunizing BALB/c mice). Meanwhile, the genetic stability and cultural character of Vero-CD150 cells were identified. Four samples were collected from clinical dogs in animal hospital .Supernatants from extracts of lung, liver, spleen were inoculate to the Vero-CD150 cells. One strain of CDV named CDV0701 was isolated by Vero-CD150 cell line and typical CPE could be seen, however CPE didn't appear on Vero cells. The stable Vero-CD150 cell line was constructed, providing an useful basis for further research works.2. Expression of CDV H gene and F gene and their use in preparation of monoclonal antibodies against CDVCDV is composed of 6 structural proteins among which Heamagglutinin protein is on the surface. H protein (H) plays a important role in CDV's pathogenicity, immunogenicity and classification. And it has a high variability. Fusion protein (F) is a protective antigen to induce neutralization antibody against CDV and to restrain symptoms when virus's generation is happening. The two proteins are the current research focus. According to the genome sequence of CDV reported in GenBank, two pairs of specific primers were designed and synthesized. Both H gene and F gene of CDV0701 strain were respectively amplified by RT-PCR, cloned and sequenced. Based on the correct results of sequencing, H and F gene were respectively inserted into the downstream of glutathione S-transferase (GST) gene of the prokaryotic expression vector pGEX-6P-1. Two recombinant vectors pGEX-H and pGEX-F were constructed and transformed into E.coLi BL21 cells respectively. Then about 52kDa GST-H fusion protein and about 56 kDa GST-F fusion protein were expressed in recombinant strain BL21 after induced by IPTG at 37℃, which was confirmed by SDS-PAGE .All the above were prepared for the preparation of monoclonal antibody against CDV.BALB/c mice were respectively immunized subcutaneously with purified fusion proteins GST-H and GST-F. Mouse spleen cells were fused with SP2/0 myeloma cells after the last immunization and hybridoma supernants were screened by indirect ELISA in time. Two hybridoma cell strains against H gene named 4A10, 2D5 and three hybridoma cell strains against F gene named 3E10, 5F9, 1G2 respectively were developed after four times of limiting dilution assay. The indirect ELISA titers of their ascites were 2.56×10~4, 2.56×10~4, 1.28×10~4, 1.28×10~4 and 2.56×10~4. The results of ELISA and Western-blot indicated that the five monoclonal antibodies were only against the responding protein but not reacted with pGEX-6P-1 protein. According to the results of IFA, the five mAb strains had specific reaction with Vero-CD150 cells infected by CDV0701. The monoclonal antibodies could be used in the research of H protein and F protein's functions and for discriminating the infection caused by CDV.