| Classical swine fever(CSF), which is caused by Classical swine fever virus(CSFV), is a highly contagious animal disease characterized by serious haemorrhage and high fever. Pigs showed high mortality and morbidity post infection. CSF was prevalent all over the world. Its outbreaks usually brought huge economic losses, and impair internal and international trade of pigs and pig’s products. It’s classified as a notifiable disease to the OIE(Office International Des Epizooties, OIE). CSFV belongs to the genus Pestivirus of the family Flaviviridae. It is an single stranded, positive-sense RNA virus with a genome length of approximately 12 kb nucleotides that encoded a polyprotein of 3898 amino acids. The translated polyprotein is processed by viral as well as cellular proteases to the mature viral proteins of four structural(C, Erns, E1, and E2) and eight nonstructural proteins(Npro, p7, NS2, NS3, NS4 A, NS4 B, NS5 A, and NS5B). The E2 envelop protein, which exposed on the outer surface of the virus, is the most immunogenic proteins responsible for inducing a protective immune response in swine and involved in the attachment and entry of CSFV. So E2 is an ideal target for therapeutic agent design.Although monoclonal antibodies against E2 glycoprotein have been developed and applied to diagnostic area, an anti-CSFV E2 glycoprotein antibody has not been reported to date and the manufacturing cost of conventional antibody was also high. To decrease the cost of diagnostic and therapeutic agent and provide new tools to study CSFV, it is necessary to discover some new antibody fragments. Nanobodies, which derived from camelid heavy-chain antibodies, is the smallest naturally occurring intact antigen binding unit. The smaller size, longer CDR3 region and existence of hydrophilic amino acids in FR2 region make them showed high affinity, high solubility, and could recognize structures inaccessible for conventional antibodies. Therefore, they are ideal candidates for therapeutic purpose.In the present study, E2 glycoprotein was used as bait to screened VHH yeast two hybrid library, which was constructed by our lab previously, using yeast two hybrid technology. The screened nanobodies were expressed using prokaryotic expression system and the reactivity with CSFV virion and E2 glycoprotein were also evaluated The main results are as follows:1. According to the CSFV sequence published on Genbank, one pair of primer was designed to amplify the E2 gene without transmembrane region. The amplified E2 gene was then successfully cloned into pGBKT7 plasmid and designed as pGBKT7-E2. Subsequently, the autoactivity and toxicity of pGBKT7-E2 were tested. Results showed that the bait had no autoactivity and toxicity. After using pGBKT7-E2 as bait to screen VHH yeast two hybrid library, eight VHH fragments were obtained for the first time.2. The eight screened VHH fragments were amplified from pGADT7-VHH and then cloned into pET28 as for expression. The optimized expression condition were induction at 30 oC for 8 h by adding 0.6 mmol/L IPTG. The E2 was also amplified and cloned into pMal-c2 X for expression. After induction at 37 oC for 5 h by adding 0.5 mmol/L IPTG, E2 was successfully expressed and purified.3. The reactivity of screened VHH and CSFV and E2 glycoprotein were tested by indirect ELISA, Western blot, Dot blot. ELISA result suggested that all the VHH could interact with CSFV and the reactivity significantly higher than negative control(P < 0.05). Western blot and Dot blot results showed that eight purified VHHs could specifically interact with E2 glycoprotein.. |
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