| Contagious Ecthyma virus (CEV) is Poxviridae which is Parapoxvirus with double stranded DNA virus. CEV is infectious disease which mainly causes proliferative lesions of infected skin and mucosa of lambs aged3-6months old. The disease spread by physical contact which is characterized by the formation of papules, vesicles, ulcers or that progress into heavy scabs on oral cavities, lips, tongues, around nostrils, breast. The disease usually spread group-occurring and the infectivity and morbidity of which is very high inside which the secondary mortality rate is up to50%. The virus spread through all of the main sheep-breeding area of Jilin Province. The disease is zoonosis, it brings huge economic loss and health risk to sheep-breeding and human beings.F1L gene is a highly conserved open reading frame with1011bp long and39kDa proteins which located in the middle of the virus genome physical map.39kDa proteins plays an important role during the ripening stages of virus. This study choose virus F1L gene as the research object,, which investigating the differences of gene sequence between this strain with another one through cloning and homologic analysis of the F1L gene of CEV isolated strain. Choosing the censervative gene fragment as the target gene for molecular biological diagnostic and exploring a practical method for restrain the spreading of CEV which could brings the important basis-and establishing the diagnosis timely. The study is divided into the following five parts.1. Isolation and identification of CEV:Using lesion tissues near by lamb’s lips which among a flock of disease lambs in Songyuan of Jilin province, Infecting newborn calf primary kidney cells self-made by lab after treatment, cells appears stable lesions by serial passage of cells. The study appraised the strain is contagious ecthyma virus and named it JLSY by the TCID50determination of the virus, type of evaluation test on virus nucleic acid, characteristic study on physical and chemical cultured, electron microscopic observation, animal regression experiment and PCR detection.2. Cloning and homologic analysis of the F1L gene of CEV from Jilin isolate:prepared newborn calf primary kidney cells by conventional methods, inoculated CEV JLSY which screened by our laboratory, observing cytopathic effect to prove Cells infected with virus, collect the virus liquid. According to the DNA sequence of the FIL gene which was published on GenBank, we designed one pair of primers to amplify F1L gene of JLSY by PCR. Link the fragment vector with pMD18-T, transformed into E.coli JM109, choose2strain virus which contain positive plasmid to keep gene sequencing before positive plasmid amplifying PCR and enzyme digestion. The amplified positive production was sequenced and compared with the sequences of several reference strains isolated in domestic. The results showed that the lowest homology with F1L gene of JLSY strains was97.6%of Strain OV/Torino, and the highest homology of Jilin strain was up to98.8%. There was little difference of F1L gene between JLSY strain and other reference strains isolated from different species and different areas in domestic. The analysis result of homology suggested that the F1L gene can be used as the target genes of the genetically engineered vaccine.3. Construction, expression and purification of CEV JLSY F1L expression vector: Designing primers which containing specific enzyme sites, amplified F1L gene, using PCR products and pET28a vector recovered by Double-enzyme Digestion of EcoRI and XhoI. Combing target fragment and vector fragment which were recovered respectively, constructing pET28a-F1L and pET28a-F1L2plasmid, and then transforming TOP10competent cells. Extracting right sequenced posrtive cloning plasmid and transforming it into BL21expression strain. Inoculating in LB culture medium in which we mix IPTF to lead the expression of protein. The expression produets detecting by SDS-PAGE, appearing protein band which is our expected goal. Concentrating soluble F1L protein which was purified by affinity chromatography, the protein concentration is0.6mg/mL.4. Establishment and preliminary application of CEV loop-mediated isothermal amplification diagnostic Methods:According to the CEV gene sequence published in GenBank, targeting a highly conserved region of the F1L gene has been developed to diagnose CEV. Sensitivity test, specificity test and replicate test of this method were promoted and then collecting clinical samples to develop preliminary application. The assay produces a ladder-like pattern of products on an agarose gel. Postive reaction tube turns green when the flurescent dye is added. The sensitivity of the LAMP assay, which was determined to be102copies of the standard plasmid, was100fold higher than PCR; furthermore, the method detection of capripox virus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I is negative;the same samples were detected for10times and the results were the same. It shows that the CEV LAMP detection method established by our study was much more sensitive specific and stable. The LAMP assay with lμL10×SYBR Green I may carry out the real-time LAMP detection and semi-quantitation experiment.30clinical samples were tested using PCR and LAMP assay, and the LAMP test results were consistent with PCR methods. The LAMP assay allows easy and rapid detection of infection with CEV and is especially applicable in a resource-limited situation.5. Establishment and preliminary application of CEV (PCR-DHPLC) diagnostic Methods: According to the gene sequence of the CEV F1L strain which published in GenBank, combining PCR with denaturing high performance liquid chromatography, designing primers with Primer Premier5.0, we designed one pair of primers to amplify F1L gene of JLSY by PCR. Analysed and detected by denaturing high performance liquid chromatography, established the detection method with PCR amplification. Sensitivity test, specificity test, stability test and clinical application test showed that the lowest copy number found by PCR-DHPLC detection method is103. The sensitivity is100higher than regular PCR electrophoresis. The specificity of our detection method is very well, which because the result of other animal virus gene DNA detection is negative and the result of CEV gene DNA with standard plasmid detection is positive. The coincidence of positive sample is100%by which using PCR-DHPLC and PCR electrophoresis detection method to detect the30clinical samples. The PCR-DHPLC detection method established by this study was much more sensitive specific and stable which could be an efficient new one for detecting the CEV. |
