本文選題：日本血吸蟲 + 未成熟蟲卵可溶性抗原(SIEA)��； 參考：《中南大學》2008年博士論文

【摘要】： 研究背景: 血吸蟲病是一種危害嚴重的人獸共患病。成熟蟲卵是血吸蟲病的主要致病因素和傳播血吸蟲病的唯一因子。因此血吸蟲病疫苗的研究主要集中于抗雌蟲生殖、抗卵胚發(fā)育方面。 近十余年來,我們課題組的研究已經(jīng)證明:未成熟蟲卵可溶性抗原(SIEA)免疫可誘導(dǎo)產(chǎn)生抗蟲卵胚胎發(fā)育和抗雌蟲生殖產(chǎn)卵的效果,SIEA誘導(dǎo)產(chǎn)生的體液免疫應(yīng)答,在抗血吸蟲病保護性免疫中起重要作用。抗SIEA免疫血清不僅與蟲卵內(nèi)胚胎結(jié)合,抑制和干擾卵胚的發(fā)育過程;而且還與雌蟲卵黃腺及緊鄰卵黃腺的腸腔內(nèi)膜組織結(jié)合,使雌蟲的生殖產(chǎn)卵功能受到影響。進一步研究發(fā)現(xiàn),SIEA26-28kDa抗原分子是誘導(dǎo)保護性體液免疫應(yīng)答的主要效應(yīng)成分之一;但是SIEA26-28kDa天然分子分離純化與批量制備困難,一直是困擾天然分子疫苗應(yīng)用的難題。因此,通過獲得高度特異性SIEA26-28kDa抗體作為探針篩選、分析并鑒定天然分子候選疫苗SIEA26-28kDa相關(guān)的編碼基因,或許是解決問題的途徑之一。近幾年來,噬菌體單鏈抗體技術(shù)的發(fā)展,為我們快速獲得具有與完整抗體相同抗原結(jié)合功能的單鏈抗體(scFv)提供了方便。 研究目的: 本研究旨在獲得與日本血吸蟲天然分子候選疫苗SIEA26-28kDa特異性結(jié)合的單鏈抗體,借此特異性單鏈抗體為探針篩選日本血吸蟲cDNA文庫,以期獲得天然分子候選疫苗相應(yīng)的編碼基因。 研究方法: (1)應(yīng)用噬菌體展示技術(shù)構(gòu)建日本血吸蟲未成熟卵單鏈抗體庫,以SIEA免疫BALB/C小鼠,提取小鼠脾臟總RNA,以總RNA為模板,逆轉(zhuǎn)錄合成cDNA第一鏈。以小鼠免疫球蛋白H鏈和L鏈可變區(qū)簡并引物,cDNA第一鏈為模板,分別擴增出H鏈和L鏈可變區(qū)基因。SOE-PCR法將VH和VL片段隨機拼接成scFv片段,然后將scFv片段克隆至pCANTAB5E載體,并電轉(zhuǎn)化至感受態(tài)TG1菌,經(jīng)輔助噬菌體M13K07拯救,獲得SIEA噬菌體展示單鏈抗體庫。測定單鏈抗體庫的庫容、重組率和多樣性。 (2)以分離純化SIEA26-28kDa天然分子抗原為靶,對單鏈抗體庫進行四輪富集,運用集落挖掘法或ELISA法篩選針對SIEA26-28kDa分子的陽性克隆。將陽性克隆感染大腸桿菌HB2151,使可溶性單鏈抗體表達。SDS-PAGE,Western blot分別鑒定單鏈抗體的表達水平、分子量以及與SIEA26-28kDa的結(jié)合活性和特異性。 (3)為了提高SIEA26-28kDa單鏈抗體的可溶性表達量,將特異性SIEA26-28kDa scFv基因序列克隆至PET32a原核表達載體,構(gòu)建PET32a/scFv原核表達質(zhì)粒;另一方面,為獲得EGFP標記的scFv,擴增增強型綠色熒光蛋白(EGFP)編碼基因,克隆至PET32a/scFv質(zhì)粒,構(gòu)建PET32a/EGFP-scFv質(zhì)粒。然后將原核重組質(zhì)粒導(dǎo)入大腸桿菌BL21(DE3)中,在大腸桿菌中誘導(dǎo)重組蛋白的表達,SDS-PAGE,Western blot分析確定單鏈抗體融合蛋白Trx-scFv和Trx-EGFP-scFv的表達水平、分子量以及與SIEA26-28kDa的結(jié)合活性和特異性。Trx-EGFP-scFv融合蛋白與日本血吸蟲成蟲和蟲卵組織切片孵育,熒光顯微鏡觀察GFP信號以評價特異性單鏈抗體的靶向性。 (4)以高表達的特異性SIEA26-28kDa單鏈抗體為探針篩選日本血吸蟲尾蚴cDNA文庫,對獲得的陽性克隆測序,通過NCBI的Blastn、Blastp程序進行核苷酸和蛋白質(zhì)水平的同源性分析。 (5)以日本血吸蟲尾蚴cDNA文庫作模板,擴增SIEA26-28kDa天然分子候選疫苗相關(guān)編碼基因的全長編碼區(qū),構(gòu)建pQE30原核重組表達質(zhì)粒。將重組表達質(zhì)粒導(dǎo)入大腸桿菌M15中,誘導(dǎo)重組蛋白的表達,SDS-PAGE,Western blot對融合蛋白表達水平,分子量及免疫反應(yīng)性進行分析。 (6)以日本血吸蟲尾蚴cDNA文庫作模板,擴增SIEA26-28kDa天然分子候選疫苗相關(guān)編碼基因的全長編碼區(qū),構(gòu)建pcDNA3真核重組表達質(zhì)粒。將昆明小鼠分為生理鹽水免疫組;pcDNA3空白質(zhì)粒免疫組:pcDNA3/SjRPS4重組質(zhì)粒免疫組;pcDNA3/SjRPL7重組質(zhì)粒免疫組。分別用空白質(zhì)粒、重組質(zhì)粒、生理鹽水免疫小鼠,免疫完畢,日本血吸蟲尾蚴攻擊感染小鼠,用減蟲率,每克肝、腸減卵率及每雌子宮內(nèi)減卵率,對目的基因動物免疫保護性效果進行評價。 研究結(jié)果: (1)構(gòu)建了日本血吸蟲未成熟蟲卵可溶性抗原單鏈抗體庫,庫容為2.27×10~7,從隨機挑選的克隆中均能擴增出大小約780bp的單鏈抗體片段;而且BstNI酶切圖譜呈多樣性。 (2)通過集落挖掘法篩選SIEA單鏈抗體庫,獲得了針對SIEA26-28kDa的特異性單鏈抗體,該特異性單鏈抗體分子量約為32kDa,但可溶性表達量較低。識別SIEA于26-28kDa的位置,條帶單一且明顯。 (3)通過雙酶切、PCR和測序鑒定,證實原核重組表達質(zhì)粒PET32a/scFv和PET32a/EGFP-scFv構(gòu)建成功�？扇苄訲rx-scFv和Trx-EGFP-scFv融合蛋白在大腸桿菌中均獲高效表達。而且融合蛋白分子量與理論預(yù)期值完全一致,同時還保存了與SIEA26-28kDa天然分子的結(jié)合活性及特異性。EGFP標記SIEA26-28kDa單鏈抗體免疫熒光定位結(jié)果顯示:熒光主要集中于未成熟蟲卵卵胚、雌蟲生殖系統(tǒng)及與生殖系統(tǒng)鄰近的腸腔組織,而在雄蟲的腸壁僅見微弱熒光。 (4)以SIEA26-28kDa單鏈抗體為探針篩選日本血吸蟲尾蚴cDNA文庫,獲得兩個編碼SIEA26-28kDa疫苗候選分子的基因:日本血吸蟲核糖體蛋白S4(SjRPS4)和日本血吸蟲核糖體蛋白L7(SjRPL7)。 (5)雙酶切、PCR和測序鑒定表明重組質(zhì)粒pQE30/SjRPS4,pQE30/SjRPL7構(gòu)建成功。SDS-PAGE和Western blot分析顯示,重組融合蛋白分子量大小與預(yù)期完全一致,并且得到了高效表達,能被特異性SIEA26-28kDa單鏈抗體、日本血吸蟲感染鼠血清識別。不能被正常鼠血清識別。 (6)雙酶切、PCR和測序鑒定表明重組質(zhì)粒pcDNA3/SjRPS4,pcDNA3/SjRPL7構(gòu)建成功。動物實驗結(jié)果表明,與對照組比較,pcDNA3/SjRPS4,pcDNA3/SjRPL7真核重組質(zhì)粒免疫組減蟲率、減卵率均明顯高于對照組,具統(tǒng)計學意義。 結(jié)論: (1)高質(zhì)量的SIEA單鏈抗體庫得以成功構(gòu)建,其庫容量大、重組率高、多樣性好,可實現(xiàn)對疫苗候選分子特異性單鏈抗體的高通量篩選。 (2)應(yīng)用集落挖掘法篩選SIEA單鏈抗體庫,快速獲得了針對SIEA26-28kDa天然分子候選疫苗的特異性單鏈抗體。 (3)SIEA26-28kDa單鏈抗體具有與完整SIEA26-28kDa抗體相同的特異性、結(jié)合力及靶向性,且易于通過基因工程實現(xiàn)大批量生產(chǎn),可完全代替完整的SIEA26-28kDa抗體,用于下一步研究。 (4)特異性SIEA26-28kDa單鏈抗體作為探針,篩選日本血吸蟲尾蚴cDNA文庫,獲得了天然分子候選疫苗SIEA26-28kDa相應(yīng)的兩個編碼基因SjRPS4和SjRPL7。 (5)SjRPS4-6×His和SjRPL7-6×His原核重組表達蛋白具有很好的免疫反應(yīng)性;pcDNA3/SjRPS4和pcDNA3/SjRPL7免疫小鼠,均獲得明顯抗血吸蟲病免疫保護性效果;減卵率普遍高于減蟲率,表明SjRPS4和SjRPL7兩基因可能主要在抗血吸蟲卵胚發(fā)育或抗雌蟲生殖方面起重要作用。為日本血吸蟲天然分子SIEA26-28kDa蛋白組分中有效的疫苗候選分子。
[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 .
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392

【引證文獻】

相關(guān)期刊論文 前1條

1 吳平;汪世平;溫志立;高冬梅;余路新;;日本血吸蟲核糖體蛋白SjRPS4基因及蛋白聯(lián)合免疫保護性價值的研究[J];中國人獸共患病學報;2010年11期

相關(guān)碩士學位論文 前4條

1 劉雙琳;日本血吸蟲SIEA26-28kDa單鏈抗體與人白介素18融合蛋白質(zhì)粒的構(gòu)建及表達[D];中南大學;2009年

2 彭微;pcDNA3.0/SjRPS4·CB多價疫苗免疫對蟲卵肉芽腫及TNF-α表達的影響[D];中南大學;2009年

3 秦永華;抗日本血吸蟲病天然分子靶基因pVAX1/SjRPS4·CB雙價疫苗與聯(lián)合免疫的研究[D];中南大學;2008年

4 吳平;日本血吸蟲核糖體蛋白SjRPS4基因及蛋白聯(lián)合免疫保護性價值的研究[D];南昌大學;2010年

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本文編號：1913996