日本血吸虫未成熟卵单链抗体库的构建、筛选及在疫苗研究中的应用

发布时间：2018-05-20 09:03

本文选题：日本血吸虫 + 未成熟虫卵可溶性抗原(SIEA)　；参考：《中南大学》2008年博士论文

【摘要】： 研究背景: 血吸虫病是一种危害严重的人兽共患病。成熟虫卵是血吸虫病的主要致病因素和传播血吸虫病的唯一因子。因此血吸虫病疫苗的研究主要集中于抗雌虫生殖、抗卵胚发育方面。近十余年来,我们课题组的研究已经证明:未成熟虫卵可溶性抗原(SIEA)免疫可诱导产生抗虫卵胚胎发育和抗雌虫生殖产卵的效果,SIEA诱导产生的体液免疫应答,在抗血吸虫病保护性免疫中起重要作用。抗SIEA免疫血清不仅与虫卵内胚胎结合,抑制和干扰卵胚的发育过程;而且还与雌虫卵黄腺及紧邻卵黄腺的肠腔内膜组织结合,使雌虫的生殖产卵功能受到影响。进一步研究发现,SIEA26-28kDa抗原分子是诱导保护性体液免疫应答的主要效应成分之一;但是SIEA26-28kDa天然分子分离纯化与批量制备困难,一直是困扰天然分子疫苗应用的难题。因此,通过获得高度特异性SIEA26-28kDa抗体作为探针筛选、分析并鉴定天然分子候选疫苗SIEA26-28kDa相关的编码基因,或许是解决问题的途径之一。近几年来,噬菌体单链抗体技术的发展,为我们快速获得具有与完整抗体相同抗原结合功能的单链抗体(scFv)提供了方便。研究目的: 本研究旨在获得与日本血吸虫天然分子候选疫苗SIEA26-28kDa特异性结合的单链抗体,借此特异性单链抗体为探针筛选日本血吸虫cDNA文库,以期获得天然分子候选疫苗相应的编码基因。研究方法: (1)应用噬菌体展示技术构建日本血吸虫未成熟卵单链抗体库,以SIEA免疫BALB/C小鼠,提取小鼠脾脏总RNA,以总RNA为模板,逆转录合成cDNA第一链。以小鼠免疫球蛋白H链和L链可变区简并引物,cDNA第一链为模板,分别扩增出H链和L链可变区基因。SOE-PCR法将VH和VL片段随机拼接成scFv片段,然后将scFv片段克隆至pCANTAB5E载体,并电转化至感受态TG1菌,经辅助噬菌体M13K07拯救,获得SIEA噬菌体展示单链抗体库。测定单链抗体库的库容、重组率和多样性。 (2)以分离纯化SIEA26-28kDa天然分子抗原为靶,对单链抗体库进行四轮富集,运用集落挖掘法或ELISA法筛选针对SIEA26-28kDa分子的阳性克隆。将阳性克隆感染大肠杆菌HB2151,使可溶性单链抗体表达。SDS-PAGE,Western blot分别鉴定单链抗体的表达水平、分子量以及与SIEA26-28kDa的结合活性和特异性。 (3)为了提高SIEA26-28kDa单链抗体的可溶性表达量,将特异性SIEA26-28kDa scFv基因序列克隆至PET32a原核表达载体,构建PET32a/scFv原核表达质粒;另一方面,为获得EGFP标记的scFv,扩增增强型绿色荧光蛋白(EGFP)编码基因,克隆至PET32a/scFv质粒,构建PET32a/EGFP-scFv质粒。然后将原核重组质粒导入大肠杆菌BL21(DE3)中,在大肠杆菌中诱导重组蛋白的表达,SDS-PAGE,Western blot分析确定单链抗体融合蛋白Trx-scFv和Trx-EGFP-scFv的表达水平、分子量以及与SIEA26-28kDa的结合活性和特异性。Trx-EGFP-scFv融合蛋白与日本血吸虫成虫和虫卵组织切片孵育,荧光显微镜观察GFP信号以评价特异性单链抗体的靶向性。 (4)以高表达的特异性SIEA26-28kDa单链抗体为探针筛选日本血吸虫尾蚴cDNA文库,对获得的阳性克隆测序,通过NCBI的Blastn、Blastp程序进行核苷酸和蛋白质水平的同源性分析。 (5)以日本血吸虫尾蚴cDNA文库作模板,扩增SIEA26-28kDa天然分子候选疫苗相关编码基因的全长编码区,构建pQE30原核重组表达质粒。将重组表达质粒导入大肠杆菌M15中,诱导重组蛋白的表达,SDS-PAGE,Western blot对融合蛋白表达水平,分子量及免疫反应性进行分析。 (6)以日本血吸虫尾蚴cDNA文库作模板,扩增SIEA26-28kDa天然分子候选疫苗相关编码基因的全长编码区,构建pcDNA3真核重组表达质粒。将昆明小鼠分为生理盐水免疫组;pcDNA3空白质粒免疫组:pcDNA3/SjRPS4重组质粒免疫组;pcDNA3/SjRPL7重组质粒免疫组。分别用空白质粒、重组质粒、生理盐水免疫小鼠,免疫完毕,日本血吸虫尾蚴攻击感染小鼠,用减虫率,每克肝、肠减卵率及每雌子宫内减卵率,对目的基因动物免疫保护性效果进行评价。研究结果: (1)构建了日本血吸虫未成熟虫卵可溶性抗原单链抗体库,库容为2.27×10~7,从随机挑选的克隆中均能扩增出大小约780bp的单链抗体片段;而且BstNI酶切图谱呈多样性。 (2)通过集落挖掘法筛选SIEA单链抗体库,获得了针对SIEA26-28kDa的特异性单链抗体,该特异性单链抗体分子量约为32kDa,但可溶性表达量较低。识别SIEA于26-28kDa的位置,条带单一且明显。 (3)通过双酶切、PCR和测序鉴定,证实原核重组表达质粒PET32a/scFv和PET32a/EGFP-scFv构建成功。可溶性Trx-scFv和Trx-EGFP-scFv融合蛋白在大肠杆菌中均获高效表达。而且融合蛋白分子量与理论预期值完全一致,同时还保存了与SIEA26-28kDa天然分子的结合活性及特异性。EGFP标记SIEA26-28kDa单链抗体免疫荧光定位结果显示:荧光主要集中于未成熟虫卵卵胚、雌虫生殖系统及与生殖系统邻近的肠腔组织,而在雄虫的肠壁仅见微弱荧光。 (4)以SIEA26-28kDa单链抗体为探针筛选日本血吸虫尾蚴cDNA文库,获得两个编码SIEA26-28kDa疫苗候选分子的基因:日本血吸虫核糖体蛋白S4(SjRPS4)和日本血吸虫核糖体蛋白L7(SjRPL7)。 (5)双酶切、PCR和测序鉴定表明重组质粒pQE30/SjRPS4,pQE30/SjRPL7构建成功。SDS-PAGE和Western blot分析显示,重组融合蛋白分子量大小与预期完全一致,并且得到了高效表达,能被特异性SIEA26-28kDa单链抗体、日本血吸虫感染鼠血清识别。不能被正常鼠血清识别。 (6)双酶切、PCR和测序鉴定表明重组质粒pcDNA3/SjRPS4,pcDNA3/SjRPL7构建成功。动物实验结果表明,与对照组比较,pcDNA3/SjRPS4,pcDNA3/SjRPL7真核重组质粒免疫组减虫率、减卵率均明显高于对照组,具统计学意义。结论: (1)高质量的SIEA单链抗体库得以成功构建,其库容量大、重组率高、多样性好,可实现对疫苗候选分子特异性单链抗体的高通量筛选。 (2)应用集落挖掘法筛选SIEA单链抗体库,快速获得了针对SIEA26-28kDa天然分子候选疫苗的特异性单链抗体。 (3)SIEA26-28kDa单链抗体具有与完整SIEA26-28kDa抗体相同的特异性、结合力及靶向性,且易于通过基因工程实现大批量生产,可完全代替完整的SIEA26-28kDa抗体,用于下一步研究。 (4)特异性SIEA26-28kDa单链抗体作为探针,筛选日本血吸虫尾蚴cDNA文库,获得了天然分子候选疫苗SIEA26-28kDa相应的两个编码基因SjRPS4和SjRPL7。 (5)SjRPS4-6×His和SjRPL7-6×His原核重组表达蛋白具有很好的免疫反应性;pcDNA3/SjRPS4和pcDNA3/SjRPL7免疫小鼠,均获得明显抗血吸虫病免疫保护性效果;减卵率普遍高于减虫率,表明SjRPS4和SjRPL7两基因可能主要在抗血吸虫卵胚发育或抗雌虫生殖方面起重要作用。为日本血吸虫天然分子SIEA26-28kDa蛋白组分中有效的疫苗候选分子。
[Abstract]:Background of Study :

Schistosoma japonicum is a kind of serious zoonotic disease . The mature insect eggs are the main pathogenic factors of schistosomiasis and the only factor to spread schistosomiasis . Therefore , the research of schistosomiasis vaccine is mainly focused on anti - female reproductive and anti - egg embryo development .

It has been proved that SIEA 26 - 28kDa antigen molecule is one of the main effective components to induce the development of anti - insect eggs , and to prevent and interfere with the development of egg embryo .

Purpose of study :

The aim of this study was to obtain a single - chain antibody which was specifically combined with the candidate vaccine SIEA26 - 28kDa of Schistosoma japonicum , whereby the specific single - chain antibody was used as the probe to screen the cDNA library of Schistosoma japonicum with a view to obtaining the corresponding coding gene of the natural molecule candidate vaccine .

Study method :

( 1 ) Using phage display technique to construct single - chain antibody library of Schistosoma japonicum , BALB / C mice were immunized with SIEA , the total RNA of mouse spleen was extracted , and the first strand of cDNA was synthesized by RT - PCR .

( 2 ) Using the purified SIEA26 - 28kDa natural molecular antigen as the target , four - wheel enrichment of single - chain antibody library was carried out . The positive clones for SIEA26 - 28kDa molecule were screened by colony - digging or ELISA . The expression level , molecular weight and binding activity and specificity of single - chain antibody were identified by SDS - PAGE and Western blot .

( 3 ) In order to increase the soluble expression of SIEA26 - 28kDa single - chain antibody , the specific SIEA26 - 28kDa scFv gene was cloned into the prokaryotic expression vector of PET 32a . The expression level , molecular weight and binding activity and specificity of the single - chain antibody fusion protein Trx - scFv and Trx - EGFP - scFv were amplified by SDS - PAGE and Western blot analysis .

( 4 ) The cDNA library of Schistosoma japonicum was screened with a high - expressed specific SIEA26 - 28kDa single - chain antibody . The obtained positive clones were sequenced , and the homology of nucleotide and protein levels was analyzed by Blasts and Blastp procedures .

( 5 ) Using the cDNA library of Schistosoma japonicum as a template , the full - length coding region of the coding gene of the SIEA26 - 28kDa natural molecule candidate vaccine was amplified , and the recombinant expression plasmid of pQE30 was constructed . The recombinant expression plasmid was introduced into the Escherichia coli M15 to induce the expression of the recombinant protein , and the expression level , molecular weight and immunoreactivity of the fusion protein were analyzed by SDS - PAGE and Western blot .

( 6 ) Construction of eukaryotic recombinant expression plasmid pcDNA3 / SjRPL7 was constructed by using the cDNA library of Schistosoma japonicum as template . The recombinant plasmid pcDNA3 / SjRPL7 was constructed by immunizing mice with pcDNA3 / SjRPS4 and pcDNA3 / SjRPL7 .

Results of the study :

( 1 ) The single - chain antibody library of immature egg - soluble antigen of Schistosoma japonicum was constructed . The library volume was 2.27 脳 10 ~ 7 , and the single - chain antibody fragment of about 780 bp was amplified from randomly selected clones .

( 2 ) The SIEA single chain antibody library was screened by colony excavation method . The specific single chain antibody against SIEA26 - 28kDa was obtained . The molecular weight of the specific single chain antibody was about 32 kDa , but the soluble expression was low . The position of SIEA at 26 - 28kDa was identified , and the band was single and obvious .

( 3 ) The fusion protein was successfully expressed in E . coli by double digestion , PCR and sequencing . Soluble Trx - scFv and Trx - EGFP - scFv fusion protein were highly expressed in E . coli . The molecular weights of soluble Trx - scFv and Trx - EGFP - scFv were fully consistent with the expected values .

( 4 ) Using SIEA26 - 28kDa single - chain antibody as probe to screen the cDNA library of Schistosoma japonicum cercariae , two genes encoding SIEA26 - 28kDa vaccine candidates were obtained : Schistosoma japonicum ribosomal protein S4 ( SjRPS4 ) and Schistosoma japonicum ribosomal protein L7 ( SjRPL7 ) .

( 5 ) The recombinant plasmid pQE30 / SjRPS4 , pQE30 / SjRPL7 was successfully constructed by double digestion , PCR and sequencing . SDS - PAGE and Western blot analysis showed that the molecular weight of the recombinant fusion protein was completely consistent with the expected .

( 6 ) The recombinant plasmid pcDNA3 / SjRPS4 and pcDNA3 / SjRPL7 were successfully constructed by double digestion , PCR and sequencing .

Conclusion :

( 1 ) the high - quality SIEA single - chain antibody library is successfully constructed , the storage capacity of the SIEA single - chain antibody library is large , the recombination rate is high , the diversity is good , and high - throughput screening of the vaccine candidate molecule - specific single - chain antibody can be realized .

( 2 ) Using colony - mining method to screen SIEA single - chain antibody library , the specific single - chain antibody against SIEA26 - 28kDa native molecule candidate vaccine was quickly obtained .

( 3 ) The SIEA26 - 28kDa single - chain antibody has the same specificity , binding force and targeting property as the intact SIEA26 - 28kDa antibody , and is easy to realize mass production through genetic engineering , and can completely replace the complete SIEA26 - 28kDa antibody for the next study .

( 4 ) Specific SIEA26 - 28kDa single - chain antibody was used as probe to screen the cDNA library of Schistosoma japonicum cercariae , and two encoding genes SjRPS4 and SjRPL7 corresponding to the natural molecule candidate SIEA26 - 28kDa were obtained .

( 5 ) SjRPS4 - 6 脳 His and SjRPL7 - 6 脳 His prokaryotic recombinant expression protein have very good immunoreactivity ; pcDNA3 / SjRPS4 and pcDNA3 / SjRPL7 immunized mice have obvious immune protective effect against schistosomiasis ; the egg reduction rate is generally higher than that of the worm reduction rate , indicating that SjRPS4 and SjRPL7 genes may play an important role in the development of Schistosoma japonicum or anti - female reproduction .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【引证文献】