Construction Of CDNA Library Of Calf Primary Oral Mucosal Epithelial Cells And Screening Of Vesicular Stomatitis Virus Receptors

Vesicular stomatitis(VS) is an acute, febrile and highly contagious infectious disease in cattle and in human which caused by vesicular stomatitis virus(VSV). The disease is characterized by vesic in the epithelium of oral mucosa, tongue, lip, papillae and coronet. The disease first occurred in the middle of United States and North America in 1821, then spreading to some countries and areas of South America, Africa, Europe and Asia. Since 1995, outbreaks of VSV had been reported in the western of United States one after another. VSV infection is frequently occurred in cattle, horses, pigs, deer and so on, but human could be infected occasionally. The mainly clinical sign of VSV infection is acute fever which is similar to Flu and Aden fever. In the summer of 2002, outbreaks of VSV were reported in Jilin province and Altay area in Xinjiang province of China. Although the infection spectrum of VSV is broad, the tropism epithelium is a common characteristics for different infected animals. However, up to now, the mechanism of tropism epithelial of VSV is not clear.In this study, we first cultured calf primary oral mucosal epithelial cells in vitro. The result indicated that we had successfully established a high-grade cultural method of calf primary oral mucosal epithelial cells (CPMBCs) by the observation of morphology, optical microscopy, ultrastructure using scanning electron microscopy and transmission electron microscopy, the identification of cytokeratin by immunohistochemistry and the examination of flow cytometry. This would establish foundation for subsequent research. We observed the morphogenesis of VSV and the changed characteristic of cellular ultrastructure using ultrathin section by collecting cells in different time after infection. The result indicated that the CPMBCs appeared cytopathic effect(CPE) after infection of VSV. We observed virions like bullet by negative staining. The result confirmed that VSV could infect primary CPMBCs. We had initially identified the process of VSV infecting CPMBCs by the observation of cellular ultrastructure. We considered that the maturity of VSV was by pullulation proliferation in cellular membrane and cytoplasm.The CPMBCs produced typical CPE after infection of VSV, so we supposed that the surface of CPMBCs should exist receptors of VSV. In order to more overall sieve the receptors of VSV, we first established T7 phage display library of CPMBCs in the research. The data showed that the library contained 1.3×107 clones, and approximately 95.8% of the library were recombinant. The titer of the amplied library was 2.4×1010 pfu/mL. The cDNA fragments longer than 300bp in length were 93% by using the PCR identification. The VSV virions after purification by sucrose gradient centrifugation were used to sieve receptors. We coated 96 ELISA plate with purificated virions, then added amplified library to carry out sieving receptors. We carried out five series sieves. At last we spread on the plate using the supernatant of the last serie. We picked out negative colonies, then extracted genome DNA from them. The cDNA inserts in these plaques were amplified by PCR using the primers. The phage DNA were sequenced , and the nucleotides were compared with the Genbank database by BLAST searches. Amino acid sequences were analyzed through ExPASy Proteomics Server and DNAStar software. About 100 clones were randomly picked from individual plaques, and their DNA sequences were amplified by PCR and analysed on agarose gel to determine the size of the inserts. The result of PCR analysis showed that 34% of the phage clones had an insert size of 400bp or longer. The sequence showed that the insert was 801bp in length and contained a 375bp open reading frame(ORF), which was predicted to encode 124 amino acids. The protein was designated CPMBCsCP1 and high homology appears between CPMBCsCP1 and the human splicing factor of 3b. The analytic result of CPMBCsCP1 protein had the characteristic of hydrophilicity, immunogenicity and good antigenicity, and biologic activity of the protein might be regulation by a variety of signals in the transmission pathway of cellular signals.This study will not only establish a foundation for the study of VSV infectious mechanism mediated by receptors, but also provide certain theory basis for the research of prevention and cure of VSV infection in our country.