Preparation and characterization of single-chain antibodies against 

Pseudomonas plecoglossicida haemolysin 

co-regulatory protein

Abstract: Visceral granulomas disease (VGD) is a bacterial disease caused by Pseudomonas plecoglossicida. It can infect a variety of aquaculture economic fishes including large yellow croaker (Larimichthys crocea), which seriously hinders the healthy and sustainable development of L. crocea aquaculture. Therefore, it is urgent to prepare a specific antibody that can quickly identify P.  plecoglossicida and can be used for research and development of rapid clinical diagnosis of VGD in L. crocea in the future.

In this study, we used P.  plecoglossicida recombinant haemolysin coregulatory protein (Hcp) as the antigen to screen the specific singlechain antibody (HcpscFv) from the rabbit natural phage scFv library using solid phase antigen screening method. After four rounds of screening and enrichment, the recovery of the fourth round was about 42 times that of the first round, indicating that the target antibody was effectively enriched. 96 phagepositive monoclones and 96 phagenegative monoclones were selected from the plate of the fourth round of elute titer determination, we coated Hcp recombinant protein on a 96well plate, and used phageELISA to detect the specificity of phage monoclones. After detecting the absorbance of phage monoclones by microplate reader and comparing the absorbance, the positive clones meeting the requirements were screened out. The obtained positive clones were amplified and the phagocytic particles for sequencing were extracted. According to the results of the obtained sequences analysis, the repeated sequences were deleted and 16 positive clones were screened out. PhageELISA assay was carried out again on 16 positive clones. We detectd and compared the absorbance of 16 positive clones. Finally 7 clones with higher specificity were selected, named A5, F11, D2, D10, G5, G8, and H12. 

According to the sequences of these seven clones, we used the Primer software to design specific primers and select appropriate restriction enzymes. Bam HⅠ and Hind Ⅲ restriction sites were added to the upstream and downstream primers of A5 and F11, and Nco Ⅰ and Not Ⅰ restriction sites were added to the upstream and downstream primers of G5, G8, D2, D10 and H12, respectively. The extracted macrophages were used as templates for PCR amplification. Then we carried out agarose gel electrophoresis experiment and recovered the target fragment. The recovered product was ligated with pEASYT vector, and the ligated product was transferred into Trans1T1 for culturing. The next day, a single colony was selected and cultured to extract plasmid pEASYTHcpScFv. We used the restriction enzymes BamH I+Hind III and Nco I+Not I to carry out double digestion on pEASYTHcpScFv and pET30a (+) respectively. The digested products were subjected to agarose gel electrophoresis, and the target fragment was recovered. And the recovered products, namely, the target fragments of pEASYTHcpScFv and expression vector pET30a (+) were ligated to construct recombinant plasmid pET30aHcpScFv.

The recombinant plasmid pET30aHcpScFv was transformed into competent Escherichia coli BL21(DE3) pLysS for small quantity induction and expression. The recombinant plasmid was induced at 30℃ for 5 h at a final concentration of 0.4 mmol·L-1 IPTG and then detected by SDSPAGE experiment. A5, F11, G5, G8, D2 and D10 all had a specific protein band at about 33 kDa. The result showed that they could be stably expressed in Escherichia coli BL21(DE3) pLysS, while H12  could not be expressed stably. 

A5, F11, G5, G8, D2, D10 were induced and expressed in large quantities. We centrifuged the bacteria to collect the bacterial precipitation and lysed it with loading buffer. After ultrasonication and centrifugation, supernatant and precipitate were collected. We lysed the precipitate with buffer B and centrifugated the liquid to obtain precipitation lysate. The target protein in supernatant and precipitation lysate was detected by SDSPAGE experiment respectively for protein solubility analysis. The results showed that F11, G8, D2 and D10 had target bands in precipitate lysates, indicating that these four strains existed as inclusion bodies. A5 and G5 had bands both in supernatant and precipitation lysate, but the protein content of precipitation lysate was significantly higher than that of supernatant, indicating that two strains mainly existed as inclusion bodies.