本文選題：日本血吸蟲(chóng) + 致弱尾蚴�。� 參考：《中南大學(xué)》2008年碩士論文

【摘要】： 研究目的 基于致弱尾蚴疫苗及天然分子疫苗高保護(hù)力的特點(diǎn),利用噬菌體抗體庫(kù)技術(shù)諸多的優(yōu)點(diǎn),構(gòu)建高效日本和血吸蟲(chóng)UV-致弱尾蚴單鏈抗體(ScFv)表達(dá)文庫(kù),并以此作為高通量的庫(kù)源篩選出針對(duì)有潛在保護(hù)力的天然分子抗原SjSCA66-68kDA的單鏈抗體,為進(jìn)一步獲取日本血吸蟲(chóng)候選疫苗天然分子SjSCA66-68kDA的相關(guān)編碼基因奠定基礎(chǔ),加快抗日本血吸蟲(chóng)病高效天然分子基因疫苗研制的步伐。 研究方法 1.利用噬菌體展示技術(shù)構(gòu)建高效日本血吸蟲(chóng)UV-照射致弱尾蚴單鏈抗體庫(kù),并從庫(kù)容量、多樣性等方面對(duì)文庫(kù)進(jìn)行鑒定。利用UV-照射日本血吸蟲(chóng)致弱尾蚴多次感染BALB/c小鼠,并將無(wú)菌UV-致弱尾蚴血清,在正常尾蚴攻擊感染(尾蚴數(shù)為40±2條)前以及感染后一周,三周,共三次尾靜脈注射轉(zhuǎn)移至BALB/c小鼠體內(nèi),45天后處死小鼠,分別計(jì)算減蟲(chóng)率和減卵率,確保其致弱尾蚴血清的高效性。在成功構(gòu)建致弱尾蚴小鼠模型的基礎(chǔ)上,提取小鼠脾臟RNA,用RT-PCR法擴(kuò)增得到全套VH和VL基因,經(jīng)重疊PCR法擴(kuò)增得到ScFv片段,將ScFv片段克隆至PCANTAB 5E載體,電轉(zhuǎn)化感受態(tài)TG1菌,經(jīng)輔助噬菌體M13K07拯救,獲得的日本血吸蟲(chóng)UV-弱尾蚴單鏈抗體庫(kù)。 2.利用建庫(kù)時(shí)所獲得的高效UV-致弱尾蚴血清與日本血吸蟲(chóng)不同發(fā)育階段的抗原進(jìn)行免疫反應(yīng),識(shí)別和選擇免疫初篩的靶分子,并對(duì)候選抗原SCA66-68天然分子免疫生化特性和序列的初步研究:通過(guò)電泳切膠、電洗脫、超濾離心和凍干等方法分離和純化SCA66-68KDa抗原,用含SCA66-68KDa抗原PAGE膠結(jié)合微量淋巴結(jié)注射法免疫新西蘭兔制備出單特異性SCA66-68KDa免疫兔血清,用ELISA和Western blot檢測(cè)和鑒定其效價(jià)和特異性;通過(guò)銀染色和PAS染色確定SCA66-68KDa抗原的化學(xué)性質(zhì);用N—端測(cè)序的方法研究SCA66-68KDa抗原的蛋白質(zhì)序列。 3.運(yùn)用分離純化的疫苗候選分子SCA66-68kDa抗原,通過(guò)抗原直接法和雙抗體夾心法聯(lián)合硝酸纖維膜富集法及菌落挖集法的組合策略篩選高通量的UV-照射致弱尾蚴單鏈抗體庫(kù),用Western blot對(duì)所獲得的特異性SCA66-68kDa單鏈抗體進(jìn)行鑒定。 研究結(jié)果: 1.UV-照射日本血吸蟲(chóng)致弱尾蚴血清被動(dòng)轉(zhuǎn)移BALB/c小鼠,可得到42.5%減蟲(chóng)率,顯著高于感染血清轉(zhuǎn)移組的減蟲(chóng)率28.0%。在成功構(gòu)建UV-照射日本血吸蟲(chóng)致弱尾蚴小鼠模型的基礎(chǔ)上,獲得了高效日本血吸蟲(chóng)UV-弱尾蚴單鏈抗體庫(kù),其的庫(kù)容量測(cè)定為1.9~*10~8,重組率100%,多樣性好。 2.實(shí)驗(yàn)發(fā)現(xiàn),建庫(kù)時(shí)的高效致弱尾蚴血清可識(shí)別血吸蟲(chóng)不同階段抗原的66-68kDa附近有共同的識(shí)別條帶,加上本室先前的研究基礎(chǔ),選擇尾蚴66-68kDa天然分子作為免疫初篩的靶分子抗原。成功分離純化了電泳純及免疫純的SCA66-68kDa抗原,并成功制備出單特異性SCA66-68KDa免疫兔血清,抗體效價(jià)高達(dá)1:12800;硝酸銀及PAS不同的染色方法來(lái)確定SCA66-68KD的化學(xué)性質(zhì)為一種蛋白類組分且是一種非糖蛋白;用N—測(cè)序的方法研究SCA66-68KDa天然分子的蛋白序列,結(jié)果存在N-端肽段封閉現(xiàn)象,提出了SCA66-68KDa天然分子的蛋白質(zhì)測(cè)序的優(yōu)化策略。 3.通過(guò)高效UV-照射致弱尾蚴血清識(shí)別了SCA66-68kDa抗原,運(yùn)用分離純化的日本血吸蟲(chóng)病疫苗候選分子SCA66-68kDa對(duì)UV-照射致弱尾蚴單鏈抗體庫(kù)進(jìn)行篩選。運(yùn)用抗原直接法和雙抗夾心硝酸纖維膜法分別經(jīng)過(guò)多輪淘洗富集,用集落挖掘法篩選致弱尾蚴單鏈抗體庫(kù):雙抗夾心法第一輪假陽(yáng)性太高,復(fù)篩沒(méi)有得到陽(yáng)性克隆;抗原直接法共獲得6個(gè)SCA66-68kDa陽(yáng)性克隆,將篩選獲得陽(yáng)性克隆轉(zhuǎn)化至Ecoli HB2151菌,誘導(dǎo)可溶性scFv表達(dá)。經(jīng)SDS-PAGE,Western blot分析,結(jié)果顯示可溶性scFv獲得了正確表達(dá),且與相應(yīng)抗原SCA66-68kDa特異性結(jié)合。結(jié)果表明通過(guò)抗原直接篩選法獲得了特異性抗SCA66-68kDa單鏈抗體。 結(jié)論: 1.首次成功構(gòu)建了高效日本血吸蟲(chóng)UV-致弱尾蚴單鏈抗體庫(kù),它的構(gòu)建彌補(bǔ)了致弱疫苗應(yīng)用的局限性,為開(kāi)拓新的疫苗發(fā)展策略和新疫苗的設(shè)計(jì)提供借鑒,也為進(jìn)一步篩選用于血吸蟲(chóng)病診斷和治療的特異性單鏈抗體、進(jìn)行抗原表位分析和疫苗研制提供了高通量的庫(kù)源。 2.以候選疫苗SjSCA66-68kDa天然分子作為初篩靶抗原,成功分離純化SjSCA66-68kDa天然分子,并對(duì)其免疫生化特性和序列進(jìn)行初步研究,為進(jìn)一步篩選致弱尾蚴單鏈抗體庫(kù)提供了理論依據(jù),也為天然分子編碼基因工程疫苗的制備奠定基礎(chǔ)。 3.成功地運(yùn)用抗原直接法聯(lián)合硝酸纖維膜富集法和集落挖掘法篩選獲得了特異性抗SCA66-68kDa單鏈抗體。SCA66-68kDa特異性ScFv的獲得在疫苗候選分子的篩選中體現(xiàn)優(yōu)勢(shì),為成功構(gòu)建有效的抗日本血吸蟲(chóng)病高效天然分子的基因工程疫苗及擴(kuò)大現(xiàn)場(chǎng)應(yīng)用奠定基礎(chǔ)。另外,在利用單鏈抗體開(kāi)展免疫診斷,免疫治療和疾病致病分子機(jī)制研究等諸多方面,顯示出廣闊的應(yīng)用前景。
[Abstract]:research objective
Based on the characteristics of the high protective ability of the weak cercariae vaccine and the natural molecular vaccine, using the advantages of the phage antibody library technology, the efficient expression library of the single chain antibody (ScFv) of the weak cercariae of Japanese and Schistosoma japonicum UV- was constructed and used as a high throughput library source to screen out the single chain of the natural molecular antigen SjSCA66-68kDA with potential protection. The antibody, which lays the foundation for further obtaining the related coding genes of the natural molecule SjSCA66-68kDA of the candidate vaccine for Schistosoma japonicum, speeds up the development of the high efficient natural molecular vaccine against schistosomiasis japonicum.
research method
1. using the phage display technique to construct the high efficient single chain antibody library of the weak cercariae caused by UV- irradiation of Schistosoma japonicum, and identify the library from the capacity and diversity of the library. UV- irradiated the weak cercariae caused by Schistosoma japonicum to infect BALB/c mice many times, and the aseptic UV- caused the weak cercariae in the normal cercariae (the number of cercariae is 40 + 2). Before and three weeks after infection, three caudal veins were injected into the BALB/c mice, and the mice were killed 45 days later to calculate the worm reduction rate and the egg reduction rate to ensure the high efficiency of the sera of the weak cercariae. On the basis of the successful construction of the mice model of the weak cercariae, the spleen RNA was extracted and the whole set of VH and VL genes were amplified by RT-PCR method. The ScFv fragment was amplified by overlapping PCR method, and the ScFv fragment was cloned to PCANTAB 5E vector, and TG1 bacteria were transformed by electricity, and the single chain antibody library of Schistosoma japonicum UV- weak cercariae was obtained by auxiliary phage M13K07.
2. the effective UV- induced weak cercariae and the antigen of different developmental stages of Schistosoma japonicum were immunized to identify and select the target molecules of the immunological screening, and the preliminary study on the immune biochemical characteristics and sequence of the candidate antigen SCA66-68 natural molecules: electrophoretic gel cutting, electric elution, ultrafiltration centrifugation and freeze drying. The SCA66-68KDa antigen was isolated and purified by the method of SCA66-68KDa antigen PAGE glue combined with microlymph node injection to immunize New Zealand rabbits with single specific SCA66-68KDa immunization. The titer and specificity were detected and identified by ELISA and Western blot, and the chemical properties of the SCA66-68KDa antigen were determined by silver staining and PAS staining; N - end was used to determine the chemical properties of the rabbit. The sequencing method was used to study the protein sequence of SCA66-68KDa antigen.
3. using the isolated and purified vaccine candidate SCA66-68kDa antigen, the single chain antibody library of high throughput UV- irradiation induced weak cercariae was screened through the combination of antigen direct method and double antibody sandwich method combined with nitric acid fibrous membrane enrichment and colony excavation method, and the specific SCA66-68kDa single chain antibody was identified by Western blot.
The results of the study:
1.UV- irradiated the weakly cercariae serum of Schistosoma japonicum to transfer BALB/c mice passively, and the 42.5% worm reduction rate was obtained, which was significantly higher than that of the infected sera group 28.0%.. On the basis of the successful construction of UV- irradiated Schistosoma japonicum induced weak cercariae, the high efficiency Japanese blood sucking worm single chain antibody library of UV- weak cercariae was obtained, and its library capacity was measured. For 1.9~*10~8, the recombination rate is 100%, and the diversity is good.
2. it was found that the high efficiency serum of cercariae in the construction of the library could identify the common bands near the 66-68kDa of different stages of Schistosoma, plus the previous research basis of this room, and selected the 66-68kDa natural molecules of cercariae as the target antigens of the immunization screening, and successfully isolated and purified the SCA66-68kDa antigens of pure electrophoresis and immuno pure. A single specific SCA66-68KDa immunized rabbit serum was successfully prepared. The antibody titer was up to 1:12800, and silver nitrate and PAS were used to determine the chemical properties of SCA66-68KD as a protein component and a non glycoprotein. The protein sequence of SCA66-68KDa natural fractions was studied by N sequencing. The results showed that the peptide segment of the N- end was closed now. Elephant, an optimization strategy for protein sequencing of SCA66-68KDa natural molecules.
3. the SCA66-68kDa antigen was identified by high efficiency UV- exposure to the weak cercariae serum, and the isolated and purified Japanese schistosomiasis vaccine candidate molecule SCA66-68kDa was used to screen the single chain antibody library of the weak cercariae induced by UV-. The single chain antibody library of the weak cercariae: the first round false positive of the double anti sandwich method was too high, and the resieve was not positive. 6 SCA66-68kDa positive clones were obtained by direct antigen method. The positive clones were screened and converted to Ecoli HB2151 bacteria to induce the expression of soluble scFv. The results showed that the soluble scFv was obtained by SDS-PAGE, Western blot analysis. The results showed that the specific anti SCA66-68kDa scFv was obtained by direct antigen screening.
Conclusion:
1. for the first time, a highly efficient single chain antibody library of Schistosoma japonicum UV- caused by Schistosoma japonicum was constructed. Its construction made up for the limitations of the application of weak vaccine. It provides reference for developing new vaccine development strategy and new vaccine design, and further screening specific single chain antibody for diagnosis and treatment of schistosomiasis, and analyzing antigen epitope analysis. And vaccine development provides a high throughput source.
2. the natural molecules of the candidate vaccine SjSCA66-68kDa were used as the primary screening target antigen, and the natural molecules of SjSCA66-68kDa were isolated and purified successfully. The preliminary study on its immune biochemical characteristics and sequences provided the theoretical basis for further screening of the single chain antibody library of the weak cercariae, and also laid the foundation for the preparation of the natural sub coding gene engineering vaccine.
3. the specificity of specific anti SCA66-68kDa single chain antibody.SCA66-68kDa specific ScFv was obtained by direct antigen direct method combined with nitric acid fibrous membrane enrichment and colony mining method to obtain the advantages of the vaccine candidate molecules, so as to successfully construct an effective gene engineering vaccine against the efficient natural molecules of schistosomiasis japonicum. In addition, the application of single chain antibody to immuno diagnosis, immunotherapy and the molecular mechanism of disease pathogenesis shows a broad application prospect.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳錦雅,楊培良,周曉紅,李華,陳曉光;應(yīng)用抗SjP38的單克隆抗體建立血吸蟲(chóng)感染的膠體金免疫層析檢測(cè)體系[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2005年05期

2 張桂筠;血吸蟲(chóng)基因工程疫苗研制策略及進(jìn)展[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));1997年01期

3 李小紅;血吸蟲(chóng)致弱疫苗的研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));2003年03期

4 朱曉華,石佑恩;血吸蟲(chóng)病全球流行病學(xué)狀況及其控制的新進(jìn)展[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));2004年03期

5 劉雪琴;汪世平;曾少華;高冬梅;何卓;;高效天然分子疫苗免疫豬血清篩選尾蚴抗原的結(jié)果分析[J];寄生蟲(chóng)與醫(yī)學(xué)昆蟲(chóng)學(xué)報(bào);2007年03期

6 吳海瑋，羅建平，，吳觀陵;用ELISA法研究血吸蟲(chóng)抗體水平時(shí)OD值標(biāo)準(zhǔn)化的方法[J];寄生蟲(chóng)與醫(yī)學(xué)昆蟲(chóng)學(xué)報(bào);1995年03期

7 岳國(guó)峰;張慧林;焦永軍;朱進(jìn);馮振卿;管曉虹;;人源日本血吸蟲(chóng)Fab抗體庫(kù)的構(gòu)建及抗獨(dú)特型抗體的篩選、鑒定[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2006年11期

8 仇鎮(zhèn)寧,馮振卿,朱昌亮,李蕓茜,管曉虹;用單克隆抗獨(dú)特型抗體NP30檢測(cè)血吸蟲(chóng)病短程抗體的研究[J];南京醫(yī)科大學(xué)學(xué)報(bào);1999年01期

9 李華,易新元,曾憲芳,陳曉光,曾慶仁,汪世平;日本血吸蟲(chóng)成蟲(chóng)67kDa分子抗原的純化及其對(duì)血吸蟲(chóng)病的診斷和療效考核[J];中國(guó)熱帶醫(yī)學(xué);2001年02期

10 劉尚林,古錦泰,張仁利,高世同;日本血吸蟲(chóng)Calpain蛋白免疫原性研究[J];中國(guó)熱帶醫(yī)學(xué);2004年04期

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