Bio-panning Of Anti-LPS VHH Against Vibrio Fluvialis To Recognize The Different Serotypes Of V. Fluvialis

Vibrio fluvialis is a kind of common conditioned pathogens, and it belongs to the vibrios. The pathogenicity of V. fluvialis is second only to Vibrio cholerae and Vibrio parahemolyticus in vibrios. V. fluvialis is also a kind of gram-negative bacteria,which is halophilic and anaerobic. This bacteria is widely distributed in seawater and harbor that contains a trace of salt. V. fluvialis can produce virulence factors related to pathogenic, such as enterotoxin material, cytotoxic, matrix metalloproteinases and hemolysin. All these virulence factors can arouse human enteritis and diarrhea which has broken out on a global scale. The infection caused by V. fluvialis is a public health problem that can't to be ignored, especially in the bad sanitary condition in the developing country. In view of the high harmfulness of V. fluvialis, therefore,establishing a rapid classification method for the serotype of V. fluvialis, has significant meaning to detect V. fluvialis quickly.Lipopolysaccharides(LPS) is a kind of bacterial endotoxin which can be only produced by gram-negative bacteria(GNB). The structure of LPS is consist of O-side chain, core polysaccharide and lipid A. The O-side chain is an important kind of specific antigen(O-antigen).The structure of lipid A and core polysaccharide is quite conservative, but the structure of O-antigen is which is most likely to mutate in LPS.The different structure of O-antigen in V. fluvialis causes the different serotype of V.Fluvialis. Up to now, the methods used for the detection of V. fluvialis are small amount. Therefore, establishing a rapid, sensitive classification method for the serotype of V. fluvialis, has important application value to study the V. fluvialis better.This study firstly inactivated the three different serotypes of V. fluvialis(Ma2598?EF85002?VF2) with high temperature. It secondly bio-pan the bacteria with using the technology of phage display and take the bacteria as antigen to pick out the nanobodies that can combine the O-antigen on the cell surface of V. fluvialis.Finally, we picked a total of 315 Phage clones to identificate the positive clones. After the phage-ELISA, 41 positive clones were selected and sent to analyze their sequences. The analytical result shows that all of the sequences are the same. Then this study constructed a pET-25b-2E3 expression vector, transfering the vector into the E. coli BL21 to express the nanobody. Then we purificated the nanobody to achieve higher purity. The nanobody that we have got was used to detect its highaffinity by phage-ELISA. The result showed that the nanobody has specificity and activity to V. fluvialis.In this study, then extracting the LPS from the three different serotypes of V.fluvialis by using the method of hot phenol water. We also purificated the LPS with enzyme hydrolysis and alcohol precipitation. Finally, we successfully obtained three different LPS from V. Fluvialis. We further selected the VHH against LPS from the camel natural phage antibody library to pick out the nanobodies that can combine the O-antigen specifically. We performed four rounds of routine screening, phage-ELISA and the analysis of the sequences. After all of these, this study worked out of eight different kinds of sequences that can specificity combined with LPS. We wish these 8positive clones could be used to classificate the three serotypes of V. fluvialis by ELISA cross reaction.