應(yīng)用噬菌體隨機肽庫篩選陰道毛滴蟲特異性B細(xì)胞表位
發(fā)布時間：2019-05-24 09:25
【摘要】：背景與目的 陰道毛滴蟲(Trichomonas vaginalis, Donne1837),是一種主要寄生于人體泌尿生殖道的鞭毛蟲,它所引起的陰道毛滴蟲病是常見的性傳播疾病之一,據(jù)WHO統(tǒng)計,全球每年約有1.8億人感染陰道毛滴蟲,同時陰道毛滴蟲的廣泛流行還增加了HIV病毒和支原體的感染率。目前對于陰道毛滴蟲病的快速診斷方法的靈敏度及特異度有限,陰道毛滴蟲疫苗的研發(fā)工作也相對滯后。本研究運用分子生物學(xué)、免疫學(xué)和生物信息學(xué)的方法技術(shù),運用噬菌體隨機7肽庫淘選陰道毛滴蟲的特異性B細(xì)胞表位,并對淘選結(jié)果進(jìn)行免疫原性,敏感性及特異性檢測,旨在為陰道毛滴蟲病的快速診斷方法及未來疫苗的研發(fā)提供理論基礎(chǔ)。 材料與方法 1.將采集到的陰道毛滴蟲臨床分離株(取自于鄭州大學(xué)第五附屬醫(yī)院檢驗科)接種于TYM培養(yǎng)基進(jìn)行連續(xù)培養(yǎng),達(dá)到純培養(yǎng)后將蟲體分別制作為全蟲抗原和凍存。 2.將全蟲抗原經(jīng)背部皮下多點注射免疫實驗用兔(購自于河南省實驗動物中心),每隔2w加強免疫2次,末次免疫后ELISA檢測抗體效價,達(dá)標(biāo)后采集、分離并純化免疫血清,并進(jìn)一步經(jīng)Western-Blot和間接免疫熒光檢測其免疫原性。 3.以純化后兔抗陰道毛滴蟲IgG對噬菌體隨機7肽庫(購自于美國NEB公司)進(jìn)行3輪的親和淘選,ELISA檢測淘選結(jié)果,并將陽性噬菌體克隆送往生物公司進(jìn)行測序,將測序結(jié)果分別與Genbank上的陰道毛滴蟲已知蛋白序列進(jìn)行比對,并在Protscale在線服務(wù)器進(jìn)行相關(guān)生物信息學(xué)分析。 4.將選取的陽性噬菌體克隆經(jīng)背部皮下多點注射免疫BALB/c小鼠(購自于河南省實驗動物中心),并同時設(shè)立陽性對照、陰性對照及溶劑對照,每隔7d免疫1次,共免疫3次。末次免疫后采集并分離免疫血清,進(jìn)行ELISA和Western-Blot檢測免疫原性,并通過間接免疫熒光試驗研究陽性噬菌體展示肽的蟲體定位。 5.建立陰道毛滴蟲小鼠皮下感染模型,每日觀察膿腫形成情況及膿液中活蟲的數(shù)量。以感染血清通過ELISA的方法檢測陽性噬菌體克隆的敏感性及特異性。 6. ELISA檢測陽性噬菌體表位肽免疫小鼠血清中細(xì)胞因子IL-4和IFN-y的水平。 結(jié)果 1.陰道毛滴蟲在48h左右達(dá)到最高生長密度,36～48h為其對數(shù)生長期,連續(xù)培養(yǎng)3代后即達(dá)到純培養(yǎng)。SDS-PAGE結(jié)果表明全蟲抗原的蛋白條帶分布在10～150kDa之間,其中30-70kDa之間的蛋白條帶最為密集。 2. ELISA檢測的結(jié)果顯示陰道毛滴蟲全蟲抗原經(jīng)免疫實驗用兔后可引起較強的免疫反應(yīng),產(chǎn)生的抗體滴度大于1：106。Western-Blot發(fā)現(xiàn)共有15條的全蟲抗原的蛋白成分可被兔免疫血清識別,比小鼠免疫血清多出3條。免疫熒光染色的結(jié)果表明蟲體可與全蟲抗原的兔免疫血清發(fā)生免疫反應(yīng),呈現(xiàn)紅色熒光。 3.經(jīng)過3輪的親和淘選,共得到14個陽性噬菌體克隆,P1～P14。P1～P14與Genbank上已知的陰道毛滴蟲蛋白序列無一級結(jié)構(gòu)的相似性。綜合噬菌體ELISA及Protscale在線服務(wù)器的生物信息學(xué)分析結(jié)果,P3、P5和P11的免疫原性相對較強。 4. ELISA結(jié)果表明P3、P5、P11免疫BALB/c小鼠后都能夠引起較強的免疫反應(yīng),差異有統(tǒng)計學(xué)意義(P0.05)。Western-Blot的結(jié)果顯示P11噬菌體克隆的免疫血清可以特異性識別全蟲抗原在42kDa左右的蛋白條帶。免疫熒光染色結(jié)果表明P3和P5免疫血清組的蟲體呈現(xiàn)出較弱的綠色熒光,而P11免疫血清組則在蟲體的膜部呈現(xiàn)出亮綠熒光。 5.皮下接種活蟲的小鼠可出現(xiàn)膿腫,在膿液中10d內(nèi)可檢出活蟲。ELISA結(jié)果表明P3、P5和P11均具有較強的特異性(P0.05),P11的敏感性為1μg/ml,P3的敏感性為2μg/ml,P5的敏感性為3μg/ml。 6. ELISA結(jié)果表明P3、P5和P11免疫完成后1w時小鼠血清中IL-4和IFN-γ的水平均高于M13組和PBS組(P0.05),IL-4的升高較為顯著,P3、P5和P11這3組之間差異無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 1.陰道毛滴蟲全蟲抗原具有良好的免疫原性,其兔免疫血清具有較高的抗體滴度。 2.應(yīng)用噬菌體隨機7肽庫淘選出14個陰道毛滴蟲模擬B細(xì)胞表位。其中P3、P5和P11具有良好的免疫原性,并具有較強的特異性及敏感性,提示這些模擬表位在研制特異性診斷試劑方面可能有潛在應(yīng)用價值。 3.P3、P5和P11免疫小鼠后可以使小鼠產(chǎn)生體液免疫及細(xì)胞免疫,以體液免疫為主。
[Abstract]:Background and Purpose Trichomonas vaginalis, Donne1837, is a kind of trichinella, which is mainly parasitic in the genitourinary tract of the human body. The trichomonas vaginalis is one of the most common sexually transmitted diseases. According to the statistics of the WHO, about 180 million people are infected with vaginal hair drops every year. The widespread popularity of Trichomonas vaginalis also increases the infection of HIV and mycoplasma The sensitivity and specificity of the rapid diagnostic method for Trichomonas vaginalis are limited, and the development of Trichomonas vaginalis vaccine is also relatively slow After using the method of molecular biology, immunology and bioinformatics, the specific B-cell epitopes of Trichomonas vaginalis were selected by using the phage-random 7-peptide library, and the immunogenicity, the sensitivity and the specific test were carried out on the result of the panning. The purpose of this study is to provide a theoretical basis for the rapid diagnosis of trichomonas vaginalis and the development of future vaccines. A. Material The method comprises the following steps of:1, inoculating the collected trichomonas vaginalis clinical isolates (taken from the Fifth Affiliated Hospital of Zhengzhou University) to a TYM culture medium for continuous culture, 2. After the last immunization, the titer of the antibody was detected by ELISA, after the last immunization, the antibody was collected and separated. The serum was purified and the serum was purified by Western-Blot and indirect immunofluorescence. 3. After purification, the rabbit anti-vaginal trichomonas IgG was used for the 3-wheel affinity panning of the phage-random 7-peptide library (available from NEB company in the United States), the panning result was detected by ELISA, and the positive phage clone was sent. sequencing the biological company, comparing the sequencing result with the known protein sequence of the Trichomonas vaginalis in Genbank, and carrying out the sequencing on the Proscale online server, Relevant bioinformatics analysis.4. The selected positive phage clones were immunized with the back subcutaneous multi-point injection of the BALB/ c mice (available from the laboratory animal center in Henan Province), and the positive control, negative control, and solvent control were also established at the same time, every 7 D. The immunosera were collected and isolated after the last immunization, and the immunogenicity was detected by ELISA and Western-Blot, and positive by indirect immunofluorescence test. 5. Establishment of a subcutaneous infection model of Trichomonas vaginalis, and daily observation of the abscess. The number of live insects in the case and in the pus is detected by the method of ELISA in the infected serum. The sensitivity and specificity of phage clone.6. The positive phage epitope peptide was detected by ELISA. factor The results showed that 1. Trichomonas vaginalis reached the highest growth density at about 48 h and 36-48 h. The results of SDS-PAGE show that the protein bands of the whole worm antigen are between 10 and 150 kDa. 2. The results of the ELISA test showed that the whole worm antigen of Trichomonas vaginalis can elicit a strong immune response after being immunized with rabbit, and the antibody titer produced is greater than 1:106. Western-Blot has found a total of 15 protein components of the whole worm antigen. It can be recognized by rabbit immune serum, and is 3 more than that of mouse immune serum. The result of immunofluorescence staining shows that the worm can be combined with the whole worm. The immune response of the rabbit immune serum of the antigen showed red fluorescence.3. After 3 rounds of affinity panning,14 positive phage clones, P1 to P14, P1 to P14 and Genban were obtained. The similarity of the first-order structure of the known Trichomonas vaginalis protein sequence on k. Bioinformatics of the comprehensive phage ELISA and the on-line server of Proscale The results showed that the immunogenicity of P3, P5 and P11 was relatively strong. The results of the immunofluorescent staining show that the insect bodies of the P3 and P5 immune serum groups show a weak green color. Light, and the P11 immune serum group showed bright green fluorescence in the membrane part of the insect body. The results of ELISA showed that P3, P5 and P11 had strong specificity (P0.05), and the sensitivity of P11 was 1. m The sensitivity of P3 was 2.mu. g/ ml, and the sensitivity of P5 was 3.mu. g/ ml. P3 There was no significant difference between the three groups of P5 and P11 (P0.05). The whole worm antigen of Trichomonas vaginalis has good immunogenicity, and the rabbit immune serum has higher antibody 2. The B-cell epitopes of 14 vagintrichomonas vaginalis were selected by using the phage-random 7-peptide library. The P3, P5 and P11 had good immunogenicity and had a strong specificity. And sensitivity, suggesting that these simulated epitopes may have potential application value in the development of specific diagnostic reagents.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.73
本文編號：2484756
[Abstract]:Background and Purpose Trichomonas vaginalis, Donne1837, is a kind of trichinella, which is mainly parasitic in the genitourinary tract of the human body. The trichomonas vaginalis is one of the most common sexually transmitted diseases. According to the statistics of the WHO, about 180 million people are infected with vaginal hair drops every year. The widespread popularity of Trichomonas vaginalis also increases the infection of HIV and mycoplasma The sensitivity and specificity of the rapid diagnostic method for Trichomonas vaginalis are limited, and the development of Trichomonas vaginalis vaccine is also relatively slow After using the method of molecular biology, immunology and bioinformatics, the specific B-cell epitopes of Trichomonas vaginalis were selected by using the phage-random 7-peptide library, and the immunogenicity, the sensitivity and the specific test were carried out on the result of the panning. The purpose of this study is to provide a theoretical basis for the rapid diagnosis of trichomonas vaginalis and the development of future vaccines. A. Material The method comprises the following steps of:1, inoculating the collected trichomonas vaginalis clinical isolates (taken from the Fifth Affiliated Hospital of Zhengzhou University) to a TYM culture medium for continuous culture, 2. After the last immunization, the titer of the antibody was detected by ELISA, after the last immunization, the antibody was collected and separated. The serum was purified and the serum was purified by Western-Blot and indirect immunofluorescence. 3. After purification, the rabbit anti-vaginal trichomonas IgG was used for the 3-wheel affinity panning of the phage-random 7-peptide library (available from NEB company in the United States), the panning result was detected by ELISA, and the positive phage clone was sent. sequencing the biological company, comparing the sequencing result with the known protein sequence of the Trichomonas vaginalis in Genbank, and carrying out the sequencing on the Proscale online server, Relevant bioinformatics analysis.4. The selected positive phage clones were immunized with the back subcutaneous multi-point injection of the BALB/ c mice (available from the laboratory animal center in Henan Province), and the positive control, negative control, and solvent control were also established at the same time, every 7 D. The immunosera were collected and isolated after the last immunization, and the immunogenicity was detected by ELISA and Western-Blot, and positive by indirect immunofluorescence test. 5. Establishment of a subcutaneous infection model of Trichomonas vaginalis, and daily observation of the abscess. The number of live insects in the case and in the pus is detected by the method of ELISA in the infected serum. The sensitivity and specificity of phage clone.6. The positive phage epitope peptide was detected by ELISA. factor The results showed that 1. Trichomonas vaginalis reached the highest growth density at about 48 h and 36-48 h. The results of SDS-PAGE show that the protein bands of the whole worm antigen are between 10 and 150 kDa. 2. The results of the ELISA test showed that the whole worm antigen of Trichomonas vaginalis can elicit a strong immune response after being immunized with rabbit, and the antibody titer produced is greater than 1:106. Western-Blot has found a total of 15 protein components of the whole worm antigen. It can be recognized by rabbit immune serum, and is 3 more than that of mouse immune serum. The result of immunofluorescence staining shows that the worm can be combined with the whole worm. The immune response of the rabbit immune serum of the antigen showed red fluorescence.3. After 3 rounds of affinity panning,14 positive phage clones, P1 to P14, P1 to P14 and Genban were obtained. The similarity of the first-order structure of the known Trichomonas vaginalis protein sequence on k. Bioinformatics of the comprehensive phage ELISA and the on-line server of Proscale The results showed that the immunogenicity of P3, P5 and P11 was relatively strong. The results of the immunofluorescent staining show that the insect bodies of the P3 and P5 immune serum groups show a weak green color. Light, and the P11 immune serum group showed bright green fluorescence in the membrane part of the insect body. The results of ELISA showed that P3, P5 and P11 had strong specificity (P0.05), and the sensitivity of P11 was 1. m The sensitivity of P3 was 2.mu. g/ ml, and the sensitivity of P5 was 3.mu. g/ ml. P3 There was no significant difference between the three groups of P5 and P11 (P0.05). The whole worm antigen of Trichomonas vaginalis has good immunogenicity, and the rabbit immune serum has higher antibody 2. The B-cell epitopes of 14 vagintrichomonas vaginalis were selected by using the phage-random 7-peptide library. The P3, P5 and P11 had good immunogenicity and had a strong specificity. And sensitivity, suggesting that these simulated epitopes may have potential application value in the development of specific diagnostic reagents.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.73
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