柑橘溃疡病菌重组单链抗体研究
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摘要
基因工程重组单克隆抗体(recombination monoclonal antibody, RMA)是90年代以来,人们利用基因工程重组技术,有目的地在基因水平上对抗体分子进行切割、拼接或修饰,或者直接合成基因序列,再将重组DNA或重组蛋白基因导入细胞表达产生的一类抗体,其化学结构特点与单克隆抗体相同,具有稳定的免疫特异性。其中单链抗体(singe chain variable fragment,ScFv)由于低或无免疫原性、分子量小、组织穿透力强,成本低,可大规模生产等特点已广泛应用于医学领域。单链抗体可以通过噬菌体展示技术或核糖体展示技术加以制备。其中,核糖体展示技术完全在体外进行,其库容量远远大于噬菌体展示文库,此外核糖体展示技术简便的建库和筛选方法,勿需选择压力,通过引入突变和重组技术来提高靶标蛋白的亲和力等优点都使核糖体展示技术显示出了诱人的发展前景。
国内外关于植物病原菌重组抗体的研究鲜见报道。只有少数应用噬菌体展示文库筛选了几种植物病原菌单链抗体,并将其应用于诊断研究中。至今无应用核糖体展示技术筛选植物病原菌单链重组抗体的报道。柑桔溃疡病(citrus bacterial canker disease, CBCD)是影响全球柑桔种植业发展的重大检疫性病害,其病原菌为地毯草黄单胞柑桔致病变种(Xanthomonas axonopodis pv. citri),是国内外重大检疫性有害生物。因抗病品种和特效药剂缺乏,美国、巴西等主要柑桔种植大国对柑桔溃疡病仍然沿用挖除病树集中烧毁的根除方法。目前我国正在实施的柑桔非疫生产区建设,强化植物检疫,严防疫害传入的监控策略,都迫切需要建立快速、特异、准确的诊断技术及防控措施。柑橘黄单孢菌(Xac)其细胞外有一层脂多糖(LPS),其有助于菌体吸附和吸收养分,增强病菌的抗逆能力,已经证实这些胞外多糖和糖蛋白是影响寄主与病原物接触识别或抑制识别和致病的生化因子。在病原菌与寄主表面接触时,识别作用决定了寄主-病原物互作性质。LPS由类脂A、核心多糖和O特异性多糖侧链(O特异性LPS)组成。其中O特异性LPS决定病原菌的种属特异性。
本研究应用核糖体展示技术建立抗柑橘溃疡病菌单链抗体文库,用O特异性LPS作为抗原筛选特异于柑橘溃疡病菌的单链抗体,并将筛选的单链抗体进行表达、纯化、研究其亲和力及特异性。本研究筛选的单链抗体可为柑橘溃疡病菌的血清学诊断提供诊断试剂,而且还可用于进一步研究柑橘溃疡病菌与寄主的初步识别机制,为柑橘溃疡病菌的生物防治提供理论依据。其主要研究结果如下:
①利用柑橘溃疡病菌细胞悬浮液免疫BALB/c小鼠,免疫后小鼠抗血清效价为2500倍左右,符合建库要求。
②提取小鼠脾细胞mRNA,构建的单链抗体文库重链大小为350bp左右,轻链为650bp左右,经linker (Gly3Ser)4连接后单链抗体大小为1.2kb左右。将单链抗体文库DNA克隆到大肠杆菌JM109中,随机挑选了9个克隆子测序表明,9条单链抗体序列都是开放阅读框,其重链分别属于VH1、VH2、VH3基因家簇,轻链属于VKⅠ、VKⅢ、VKⅣ亚基因家簇。每个单链抗体的互补决定区(CDRs)都为不同的CDR,其中氨基酸序列变化多样,说明构建的单链抗体文库多样性好,适合于进一步进行单链抗体的筛选。
③将构建的单链抗体文库进行体外转录和体外翻译后,产生了单链抗体-核糖体-mRNA(ARM)三联复合体文库。用柑橘溃疡病菌O特异性脂多糖亲和筛选翻译产生的ARM三联复合体,洗涤后,将保留的ARM复合体解离,释放mRNA,并对mRNA进行了反转录PCR扩增得到筛选后的单链抗体DNA文库,然后重复体外转录-体外翻译-亲和筛选-RT-PCR扩增过程,得到反复筛选几轮的单链抗体DNA文库。结果显示第一轮核糖体展示后回收的mRNA量非常少,分光光度计已测不出其浓度,反转录RCR后,扩增得到的条带非常弱,说明在原始未筛选的抗体文库中,能与柑橘溃疡病菌O特异性脂多糖作用的单链抗体数量较少。经过三轮筛选后,得到的mRNA量逐渐增多,经RT-PCR后,产生了比较亮的扩增条带。说明在核糖体展示过程中,抗原阳性的单链抗体得到了富集。
④将未经过筛选的原始单链抗体文库DNA和经过三轮筛选的单链抗体文库DNA与噬菌体表达载体pCANTAB5E相连接后,转入大肠杆菌TG1中小量表达。表达后用间接ELISA测定单链抗体与抗原的结合活性。结果表明从未经过筛选的原始单链抗体文库中随机挑取的60个克隆子表达产物与柑橘溃疡病菌O特异性脂多糖几乎没有结合能力;而从经过三轮筛选后的单链抗体文库中挑取的60个克隆子中有30%与单链抗体有较好的结合能力。从三轮筛选后的单链抗体文库中共挑取了180个克隆子,用间接ELISA法初筛到60株抗原阳性的单链抗体;然后用生物传感技术(biosensor, biacore)对筛选的抗原阳性的单链抗体进行了复筛,筛选了3株高亲和力的单链抗体(GX13、GX44和GX95)以用于下一步的表达鉴定。
⑤将筛选的高亲和力抗原阳性的克隆子从大肠杆菌TG1中转入高表达菌株HB2151中进行可溶性表达。并优化了表达条件。优化的表达条件如下:将单链抗体单克隆子接种于5 ml 2×YTAG中,30℃250 r/min培养过夜;次日将1ml过夜培养液加入50 ml 2×YTAG (2×YT培养基中含2%葡萄糖,100μg/ml氨苄青霉素)中,30℃250 r/min培养至OD_(600)为0.6-0.8;3500 r/min离心20 min,弃去上清;50 ml 2×YTAI(2×YT培养基中含1 mM IPTG,100μg/ml氨苄青霉素)重悬沉淀,30℃250 r/min培养7 h;3500 r/min离心20 min,沉淀用0.5 ml冰冷的1×TES (0.2 M Tris–HCl [pH 8.0], 0.5 mM EDTA, 0.5 M sucrose)重悬,再加入0.75 ml冰冷的1/4×TES,Vortex重悬,冰上孵育30 min,高速离心l0 min,留取上清,-20℃保存,此上清中含有分泌至细胞周质中的(perplasmic extract)抗体。
⑥单链抗体表达后,其表达产物主要集中于细胞周质提取物中,具有抗体活性。将浓缩的周质提取物进行SDS-PAGE电泳,显示在32 kDa处有一蛋白条带产生。将表达产物纯化后进行SDS-PAGE显示,在32 kDa同样有一蛋白条带产生。为了进一步验证表达的蛋白即目的蛋白,将表达产物进行了Western blot杂交,结果显示,与纯化后的电泳结果一致,在32 kDa处有单一的条带产生,说明表达纯化的蛋白即目的蛋白。
⑦将筛选的抗原阳性单链抗体进行了特性研究。单链抗体(GX95、GX44、GX13)特异性强、亲和力高。其与柑橘溃疡病菌近源种Xanthomonas. oryzae pv. oryzae (Xooc); Xanthomonas. campestris pv. campestri (Xcc); Xanthomonas. oryzae pv. oryzicola (Xoc);及从柑橘叶片上分离的10种腐生黄单孢菌及Bacillus subtilis; E. coli都没有交叉反应。Biacore分析其亲和力表明,单链抗体GX95、GX44和GX13的亲和常数分别为1.98×10~(10) M~(-1)、1.89×10~(10) M~(-1)、3.43×10~(10) M~(-1)。⑧对筛选的单链抗体进行了测序。用DNAplot软件分析单链抗体序列,结果表明:单链抗体GX44和GX13重链分别属于VH1基因家簇,GX95重链属于VH3基因家簇;GX44和GX13轻链属于Vk IV亚基因家簇,GX95轻链属于Vk III亚基因家簇。用Vector NTI软件对筛选的单链抗体的序列同源性进行了分析,表明GX44和GX13重链有89.67%的同源性,GX95和GX13具有92.53%的同源性。
Recombination monoclonal antibodies (RMA) are artificial constructions produced by recombining the antibody molecule or synthesizing the gene sequence with gene recombination technology and then transforming the antibody DNA into cells for expression. The RMAs keep the binding properties of classical antibody. Among the RMAs, the single chain variable fragments (ScFvs) have been widely applicated to medical researches because of its reduced immunogenicity, small molecule, high tissue penetration, low production cost. ScFvs can be made by phage display or ribosome display technology, and the former have some obvious advantages than the later, such as simple method for construction of library and selection of antibody, no panning pressure, and improved antibody affinity by introducing mutation.
There have been few reports about recombination monoclonal antibodies against phytopathogen except several reports about selection of ScFvs for diagnosing research by phage display. There have been no reports about selection of ScFvs against phytopathogen by ribosome display. Citrus canker caused by the bacterial pathogen Xanthomonas axonopodis pv. citri (Xac), a gram-negative bacterium, is a severe bacterial disease of most commercial citrus species and cultivars around the world, as well as some citrus relatives. The pathogen is the target of quarantine efforts abroad and domestics, then the development of rapid and reliable procedures for diagnosis and control of this pathogen has been an important priority. Lippolysaccharide (LPS) is a major component of outer membrane of gram-negative bacteria, and is suspected to be an important molecule for adhesion to and infection of plants. The LPS consists of O-specific side chains, core polysaccharide and lipid A, in which the O-specific side chain determines species specificity.
For the study presented here, the O-specific LPS of Xac was used as an antigen to pan specific ScFvs against Xac by ribosome display. The selected ScFvs can be further applied to development of diagnostic reagent of Xac and research on the interaction between Xac pathogen and citrus during the phase of initial attachment-infection process. The main results are as follows:
①The Xac cell suspensions were used to immune the BALB/c mice and the titer of immunized mouse was 2500-fold.
②The mRNA of mouse spleen cell was extracted for construction antibody library. The size of amplificated VH was 350 bp and that of VL was 650 bp. The VH and VL were ligated with the linker (Gly3Ser)4 and the assembled ScFvs were of expected 1.2 kb size. The ScFv DNA was cloned into PMD18-T, and nine clones were selected randomly for sequencing. The VH and Vk gene families of the ScFvs were designated based on Werner Muller’s database (DNAPLOT software). The heavy chains of those belonged to the VH、VH2 and VH3 and the light chains belonged to the VKⅠ, VKⅢand Vk IV subgroup, respectively. Each CDR of ScFvs was different and the amine acid sequences of the CDRs were diversity. It showed that the constructed library was diversity and could be used to selection of specific ScFvs.
③In each round of ribosome display, the VH/k-DNA library was used for in vitro transcription with T7 RNA polymerase and the mRNA transcripts was translated in rabbit reticulocyte lysate system to produce antibody–ribosome–mRNA (ARM) complexes. The ARM complexes were then added to the O-specific LPS-coated microtiter plates and incubated. After washing, the retained ARM complexes were dissociated and the released mRNA was recovered by RT-PCR. The progress of panning was monitored by examining the intensity of RT-PCR products on agarose gel. By the end of the first round, only a weak DNA band was visible. The quantity of RT-PCR products continually increased during the next rounds of panning. Based on the result of RT-PCR, enrichment of specific ScFvs was clearly confirmed.
④DNA outputs from the unselected and the third selected antibody library were ligated with express vector pCANTAB-5E and then cloned into E.coli TG1 for soluble ScFvs expression. After transformation, 120 clones from the two libraries were isolated randomly and soluble proteins of these clones were expressed. The periplasmic extracts from these clones were tested for production of antigen-specific ScFvs by indirect ELISA. The result showed that all the isolated clones from the unselected library had little binding activity to O-specific LPS, but about 30% of the clones from the third selected library had a good conjugation activity. It also indicated that the ScFvs against O-specific LPS were enriched by ribosome display. Approximately 180 clones from the third selected library were analyzed by ELISA and about 60 clones of those reacted positively with O-specific LPS. Among them, 3 clones showed high affinity by Biacore analysis.
⑤The selected antigen-positive ScFvs were transferred into HB2151 for high soluble expression. Briefly, 1ml overnight culture was added to 50 ml of freshly prepared 2×YT medium containing 100μg/ml ampicillin and 2% (w/v) glucose and cultured at 30℃with shaking at 250 r/min until they reached an absorbance of 0.6-0.8 at 600 nm. Then the cells were centrifuged and the supernatants were removed. The sediment cells were resuspended in 50 ml of freshly prepared 2×YT containing 100μg/ml ampicillin and 1 mM IPTG, and then incubated for 7 h at 30℃with shaking at 250 r/min. The cells were pelleted and resuspended in 0.5 ml ice-cold 1×TES buffer (0.2 M Tris–HCl [pH 8.0], 0.5 mM EDTA, 0.5 M sucrose) and 0.75 ml icecold 1/4×TES buffer. After incubation on ice for 30 min, the cells were pelleted by centrifugation at 10, 000 r/min for 10 min and the supernatant was retained as periplasmic extracts containing the soluble ScFvs.
⑥The TCA-concentrated periplamic extract and purified ScFvs were run on a denaturing polyacrylamide gel and about 32kDa protein band was produced. The TCA-concentrated periplamic extract on the denaturing polyacrylamide gel were transferred to PVDF membrane for western blot analysis. A band at 32kDa was apparent.
⑦The specificity of the three selected ScFvs (GX95, GX44 and GX13) was tested by indirect ELISA and Biacore analysis. The Xac and other bacteria (Xanthomonas. oryzae pv. oryzae, Xooc; Xanthomonas. campestris pv. campestri, Xcc; Xanthomonas. oryzae pv. oryzicola, Xoc; B. subtilis; E. coli and 10 saprophytic xanthomonads isolated from leaves of healthy citrus) were tested. The results showed that the three ScFvs had good activity with Xac, while no cross reaction with the other bacteria. The equilibrium constant (KA) determined by BIAcore analysis for GX13, GX44 and GX95 were 1.98×10~(10) M~(-1), 1.89×10~(10) M~(-1) and 3.43×10~(10) M~(-1), respectively.
⑧The three ScFv GX95, GX44 and GX13 were sequenced. Using the DNA sequences, the VH and Vk gene families of the ScFvs were designated based on Werner Muller’s database (DNAPLOT software). The heavy chain of ScFv GX44 and GX13 belonged to the VH1 gene family and that of GX95 belonged to the VH3 gene family. The light chain of GX44 and GX13 belonged to the Vk IV subgroup and that of GX95 belonged to the Vk III subgroup. Sequencing alignment using the Vector NTI software showed that the VH of GX44 and GX13 shared 89.67% homology and the Vk of GX95 and GX13 shared 92.53% homology.
国内外关于植物病原菌重组抗体的研究鲜见报道。只有少数应用噬菌体展示文库筛选了几种植物病原菌单链抗体,并将其应用于诊断研究中。至今无应用核糖体展示技术筛选植物病原菌单链重组抗体的报道。柑桔溃疡病(citrus bacterial canker disease, CBCD)是影响全球柑桔种植业发展的重大检疫性病害,其病原菌为地毯草黄单胞柑桔致病变种(Xanthomonas axonopodis pv. citri),是国内外重大检疫性有害生物。因抗病品种和特效药剂缺乏,美国、巴西等主要柑桔种植大国对柑桔溃疡病仍然沿用挖除病树集中烧毁的根除方法。目前我国正在实施的柑桔非疫生产区建设,强化植物检疫,严防疫害传入的监控策略,都迫切需要建立快速、特异、准确的诊断技术及防控措施。柑橘黄单孢菌(Xac)其细胞外有一层脂多糖(LPS),其有助于菌体吸附和吸收养分,增强病菌的抗逆能力,已经证实这些胞外多糖和糖蛋白是影响寄主与病原物接触识别或抑制识别和致病的生化因子。在病原菌与寄主表面接触时,识别作用决定了寄主-病原物互作性质。LPS由类脂A、核心多糖和O特异性多糖侧链(O特异性LPS)组成。其中O特异性LPS决定病原菌的种属特异性。
本研究应用核糖体展示技术建立抗柑橘溃疡病菌单链抗体文库,用O特异性LPS作为抗原筛选特异于柑橘溃疡病菌的单链抗体,并将筛选的单链抗体进行表达、纯化、研究其亲和力及特异性。本研究筛选的单链抗体可为柑橘溃疡病菌的血清学诊断提供诊断试剂,而且还可用于进一步研究柑橘溃疡病菌与寄主的初步识别机制,为柑橘溃疡病菌的生物防治提供理论依据。其主要研究结果如下:
①利用柑橘溃疡病菌细胞悬浮液免疫BALB/c小鼠,免疫后小鼠抗血清效价为2500倍左右,符合建库要求。
②提取小鼠脾细胞mRNA,构建的单链抗体文库重链大小为350bp左右,轻链为650bp左右,经linker (Gly3Ser)4连接后单链抗体大小为1.2kb左右。将单链抗体文库DNA克隆到大肠杆菌JM109中,随机挑选了9个克隆子测序表明,9条单链抗体序列都是开放阅读框,其重链分别属于VH1、VH2、VH3基因家簇,轻链属于VKⅠ、VKⅢ、VKⅣ亚基因家簇。每个单链抗体的互补决定区(CDRs)都为不同的CDR,其中氨基酸序列变化多样,说明构建的单链抗体文库多样性好,适合于进一步进行单链抗体的筛选。
③将构建的单链抗体文库进行体外转录和体外翻译后,产生了单链抗体-核糖体-mRNA(ARM)三联复合体文库。用柑橘溃疡病菌O特异性脂多糖亲和筛选翻译产生的ARM三联复合体,洗涤后,将保留的ARM复合体解离,释放mRNA,并对mRNA进行了反转录PCR扩增得到筛选后的单链抗体DNA文库,然后重复体外转录-体外翻译-亲和筛选-RT-PCR扩增过程,得到反复筛选几轮的单链抗体DNA文库。结果显示第一轮核糖体展示后回收的mRNA量非常少,分光光度计已测不出其浓度,反转录RCR后,扩增得到的条带非常弱,说明在原始未筛选的抗体文库中,能与柑橘溃疡病菌O特异性脂多糖作用的单链抗体数量较少。经过三轮筛选后,得到的mRNA量逐渐增多,经RT-PCR后,产生了比较亮的扩增条带。说明在核糖体展示过程中,抗原阳性的单链抗体得到了富集。
④将未经过筛选的原始单链抗体文库DNA和经过三轮筛选的单链抗体文库DNA与噬菌体表达载体pCANTAB5E相连接后,转入大肠杆菌TG1中小量表达。表达后用间接ELISA测定单链抗体与抗原的结合活性。结果表明从未经过筛选的原始单链抗体文库中随机挑取的60个克隆子表达产物与柑橘溃疡病菌O特异性脂多糖几乎没有结合能力;而从经过三轮筛选后的单链抗体文库中挑取的60个克隆子中有30%与单链抗体有较好的结合能力。从三轮筛选后的单链抗体文库中共挑取了180个克隆子,用间接ELISA法初筛到60株抗原阳性的单链抗体;然后用生物传感技术(biosensor, biacore)对筛选的抗原阳性的单链抗体进行了复筛,筛选了3株高亲和力的单链抗体(GX13、GX44和GX95)以用于下一步的表达鉴定。
⑤将筛选的高亲和力抗原阳性的克隆子从大肠杆菌TG1中转入高表达菌株HB2151中进行可溶性表达。并优化了表达条件。优化的表达条件如下:将单链抗体单克隆子接种于5 ml 2×YTAG中,30℃250 r/min培养过夜;次日将1ml过夜培养液加入50 ml 2×YTAG (2×YT培养基中含2%葡萄糖,100μg/ml氨苄青霉素)中,30℃250 r/min培养至OD_(600)为0.6-0.8;3500 r/min离心20 min,弃去上清;50 ml 2×YTAI(2×YT培养基中含1 mM IPTG,100μg/ml氨苄青霉素)重悬沉淀,30℃250 r/min培养7 h;3500 r/min离心20 min,沉淀用0.5 ml冰冷的1×TES (0.2 M Tris–HCl [pH 8.0], 0.5 mM EDTA, 0.5 M sucrose)重悬,再加入0.75 ml冰冷的1/4×TES,Vortex重悬,冰上孵育30 min,高速离心l0 min,留取上清,-20℃保存,此上清中含有分泌至细胞周质中的(perplasmic extract)抗体。
⑥单链抗体表达后,其表达产物主要集中于细胞周质提取物中,具有抗体活性。将浓缩的周质提取物进行SDS-PAGE电泳,显示在32 kDa处有一蛋白条带产生。将表达产物纯化后进行SDS-PAGE显示,在32 kDa同样有一蛋白条带产生。为了进一步验证表达的蛋白即目的蛋白,将表达产物进行了Western blot杂交,结果显示,与纯化后的电泳结果一致,在32 kDa处有单一的条带产生,说明表达纯化的蛋白即目的蛋白。
⑦将筛选的抗原阳性单链抗体进行了特性研究。单链抗体(GX95、GX44、GX13)特异性强、亲和力高。其与柑橘溃疡病菌近源种Xanthomonas. oryzae pv. oryzae (Xooc); Xanthomonas. campestris pv. campestri (Xcc); Xanthomonas. oryzae pv. oryzicola (Xoc);及从柑橘叶片上分离的10种腐生黄单孢菌及Bacillus subtilis; E. coli都没有交叉反应。Biacore分析其亲和力表明,单链抗体GX95、GX44和GX13的亲和常数分别为1.98×10~(10) M~(-1)、1.89×10~(10) M~(-1)、3.43×10~(10) M~(-1)。⑧对筛选的单链抗体进行了测序。用DNAplot软件分析单链抗体序列,结果表明:单链抗体GX44和GX13重链分别属于VH1基因家簇,GX95重链属于VH3基因家簇;GX44和GX13轻链属于Vk IV亚基因家簇,GX95轻链属于Vk III亚基因家簇。用Vector NTI软件对筛选的单链抗体的序列同源性进行了分析,表明GX44和GX13重链有89.67%的同源性,GX95和GX13具有92.53%的同源性。
Recombination monoclonal antibodies (RMA) are artificial constructions produced by recombining the antibody molecule or synthesizing the gene sequence with gene recombination technology and then transforming the antibody DNA into cells for expression. The RMAs keep the binding properties of classical antibody. Among the RMAs, the single chain variable fragments (ScFvs) have been widely applicated to medical researches because of its reduced immunogenicity, small molecule, high tissue penetration, low production cost. ScFvs can be made by phage display or ribosome display technology, and the former have some obvious advantages than the later, such as simple method for construction of library and selection of antibody, no panning pressure, and improved antibody affinity by introducing mutation.
There have been few reports about recombination monoclonal antibodies against phytopathogen except several reports about selection of ScFvs for diagnosing research by phage display. There have been no reports about selection of ScFvs against phytopathogen by ribosome display. Citrus canker caused by the bacterial pathogen Xanthomonas axonopodis pv. citri (Xac), a gram-negative bacterium, is a severe bacterial disease of most commercial citrus species and cultivars around the world, as well as some citrus relatives. The pathogen is the target of quarantine efforts abroad and domestics, then the development of rapid and reliable procedures for diagnosis and control of this pathogen has been an important priority. Lippolysaccharide (LPS) is a major component of outer membrane of gram-negative bacteria, and is suspected to be an important molecule for adhesion to and infection of plants. The LPS consists of O-specific side chains, core polysaccharide and lipid A, in which the O-specific side chain determines species specificity.
For the study presented here, the O-specific LPS of Xac was used as an antigen to pan specific ScFvs against Xac by ribosome display. The selected ScFvs can be further applied to development of diagnostic reagent of Xac and research on the interaction between Xac pathogen and citrus during the phase of initial attachment-infection process. The main results are as follows:
①The Xac cell suspensions were used to immune the BALB/c mice and the titer of immunized mouse was 2500-fold.
②The mRNA of mouse spleen cell was extracted for construction antibody library. The size of amplificated VH was 350 bp and that of VL was 650 bp. The VH and VL were ligated with the linker (Gly3Ser)4 and the assembled ScFvs were of expected 1.2 kb size. The ScFv DNA was cloned into PMD18-T, and nine clones were selected randomly for sequencing. The VH and Vk gene families of the ScFvs were designated based on Werner Muller’s database (DNAPLOT software). The heavy chains of those belonged to the VH、VH2 and VH3 and the light chains belonged to the VKⅠ, VKⅢand Vk IV subgroup, respectively. Each CDR of ScFvs was different and the amine acid sequences of the CDRs were diversity. It showed that the constructed library was diversity and could be used to selection of specific ScFvs.
③In each round of ribosome display, the VH/k-DNA library was used for in vitro transcription with T7 RNA polymerase and the mRNA transcripts was translated in rabbit reticulocyte lysate system to produce antibody–ribosome–mRNA (ARM) complexes. The ARM complexes were then added to the O-specific LPS-coated microtiter plates and incubated. After washing, the retained ARM complexes were dissociated and the released mRNA was recovered by RT-PCR. The progress of panning was monitored by examining the intensity of RT-PCR products on agarose gel. By the end of the first round, only a weak DNA band was visible. The quantity of RT-PCR products continually increased during the next rounds of panning. Based on the result of RT-PCR, enrichment of specific ScFvs was clearly confirmed.
④DNA outputs from the unselected and the third selected antibody library were ligated with express vector pCANTAB-5E and then cloned into E.coli TG1 for soluble ScFvs expression. After transformation, 120 clones from the two libraries were isolated randomly and soluble proteins of these clones were expressed. The periplasmic extracts from these clones were tested for production of antigen-specific ScFvs by indirect ELISA. The result showed that all the isolated clones from the unselected library had little binding activity to O-specific LPS, but about 30% of the clones from the third selected library had a good conjugation activity. It also indicated that the ScFvs against O-specific LPS were enriched by ribosome display. Approximately 180 clones from the third selected library were analyzed by ELISA and about 60 clones of those reacted positively with O-specific LPS. Among them, 3 clones showed high affinity by Biacore analysis.
⑤The selected antigen-positive ScFvs were transferred into HB2151 for high soluble expression. Briefly, 1ml overnight culture was added to 50 ml of freshly prepared 2×YT medium containing 100μg/ml ampicillin and 2% (w/v) glucose and cultured at 30℃with shaking at 250 r/min until they reached an absorbance of 0.6-0.8 at 600 nm. Then the cells were centrifuged and the supernatants were removed. The sediment cells were resuspended in 50 ml of freshly prepared 2×YT containing 100μg/ml ampicillin and 1 mM IPTG, and then incubated for 7 h at 30℃with shaking at 250 r/min. The cells were pelleted and resuspended in 0.5 ml ice-cold 1×TES buffer (0.2 M Tris–HCl [pH 8.0], 0.5 mM EDTA, 0.5 M sucrose) and 0.75 ml icecold 1/4×TES buffer. After incubation on ice for 30 min, the cells were pelleted by centrifugation at 10, 000 r/min for 10 min and the supernatant was retained as periplasmic extracts containing the soluble ScFvs.
⑥The TCA-concentrated periplamic extract and purified ScFvs were run on a denaturing polyacrylamide gel and about 32kDa protein band was produced. The TCA-concentrated periplamic extract on the denaturing polyacrylamide gel were transferred to PVDF membrane for western blot analysis. A band at 32kDa was apparent.
⑦The specificity of the three selected ScFvs (GX95, GX44 and GX13) was tested by indirect ELISA and Biacore analysis. The Xac and other bacteria (Xanthomonas. oryzae pv. oryzae, Xooc; Xanthomonas. campestris pv. campestri, Xcc; Xanthomonas. oryzae pv. oryzicola, Xoc; B. subtilis; E. coli and 10 saprophytic xanthomonads isolated from leaves of healthy citrus) were tested. The results showed that the three ScFvs had good activity with Xac, while no cross reaction with the other bacteria. The equilibrium constant (KA) determined by BIAcore analysis for GX13, GX44 and GX95 were 1.98×10~(10) M~(-1), 1.89×10~(10) M~(-1) and 3.43×10~(10) M~(-1), respectively.
⑧The three ScFv GX95, GX44 and GX13 were sequenced. Using the DNA sequences, the VH and Vk gene families of the ScFvs were designated based on Werner Muller’s database (DNAPLOT software). The heavy chain of ScFv GX44 and GX13 belonged to the VH1 gene family and that of GX95 belonged to the VH3 gene family. The light chain of GX44 and GX13 belonged to the Vk IV subgroup and that of GX95 belonged to the Vk III subgroup. Sequencing alignment using the Vector NTI software showed that the VH of GX44 and GX13 shared 89.67% homology and the Vk of GX95 and GX13 shared 92.53% homology.
引文
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