抗杀香鱼假单胞菌溶血素共调节蛋白单链抗体的制备及特性分析

Preparation and characterization of single-chain antibodies against Pseudomonas plecoglossicida haemolysin co-regulatory protein

杀香鱼假单胞菌(Pseudomonas plecoglossicida)是大黄鱼(Larimichthys crocea)内脏白点病的主要致病菌,为制备能识别杀香鱼假单胞菌的特异性抗体,采用固相化抗原筛选方法,以杀香鱼假单胞菌重组溶血素共调节蛋白为抗原,从兔天然噬菌体scFv文库中筛选出特异性的单链抗体(scFv);通过构建含有针对杀香鱼假单胞菌单链抗体(scFv)的重组质粒,对筛选出的scFv进行原核诱导表达。通过4轮淘选,共获得16株针对杀香鱼假单胞菌溶血素共调节蛋白的单链抗体(scFv),通过亲和力分析,最终筛选出7株特异性较高的单链抗体(scFv)进行原核表达;成功构建了含有抗杀香鱼假单胞菌溶血素共调节单链抗体(scFv)的重组质粒pET-30a-Hcp-scFv,转化到大肠杆菌表达菌株中,其中6个重组质粒可稳定表达,经过蛋白质可溶性分析和Western Blot鉴定,目的蛋白主要以包涵体的形式存在。研究结果为快速制备大量抗杀香鱼假单胞菌抗体提供了一种简便的方法,为今后大黄鱼内脏白点病的临床快速诊断奠定了基础。

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 HⅠand HindⅢrestriction sites were added to the upstream and downstream primers of A5 and F11,and NcoⅠand NotⅠrestriction 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-30 a(+)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-30 a(+)were ligated to construct recombinant plasmid pET-30 a-Hcp-ScFv.The recombinant plasmid pET-30 a-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.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 Ni-Denature-urea buffer.After ultrasonication and centrifugation,we collected supernatant,namely precipitation lysate,and the target protein bands were successfully observed by SDS-PAGE experiment.The protein solution was purified by Ni-IDA-6 FF pre-packed gravity column,and relatively clear target protein bands were observed at the expected position by SDS-PAGE experiment.Western Blot experiment was carried out to further check whether the target protein structure was intact after purification,using mouse primary antibody corresponding to His-tag on the target protein and goat anti-mouse secondary antibody.The results showed that obvious brown bands could be observed on the PVDF membrane at the position of 35 kDa,which was consistent with the position of the bands in the SDS-PAGE experiment,indicating that the target protein had a specific reaction with His-tag mouse monoclonal antibody.The result proved that the recombinant protein was structurally intact after purification,which also indicated that pET-30 a-Hcp-ScFv against P.plecoglossicida recombinant Hcp could be effectively expressed in Escherichia coli BL21(DE3)pLysS.The result of this study provided a simple and rapid method for the rapid preparation of a large number of specific antibodies against P.plecoglossicida recombinant Hcp.We used the solid phase antigen screening method and the principle of specific binding of antigens and antibodies to screen the specific scFv from the rabbit natural phage scFv library.Then the specific scFv was ligated with pET-30 a(+)to construct a recombinant plasmid.In order to obtain the recombinant protein,we transferred the recombinant plasmid into E.coil BL21(DE3)pLySs for induction and expression.Finally,the target antibody was obtained by purifying the recombinant protein.The result provides a scientific basis for the clinical rapid diagnosis technology of VGD in L.crocea in the future.