【作者】 王莉；

【导师】 崔晶；

【作者基本信息】 郑州大学 ， 病原生物学， 2014， 博士

【摘要】 旋毛虫病(trichinellosis)主要因生食或半生食含有旋毛虫(Trichinella)幼虫的猪肉或其它动物肉类而感染,是一种呈全球性分布的食源性人兽共患寄生虫病。由于旋毛虫病无特异性的症状和体征,因此,临床上对本病不易进行及时、正确的诊断。目前,该病的确诊主要依靠肌肉活检和高特异性的免疫学诊断方法,但前者取决于肌肉样本的大小及感染程度,对于轻度和早期感染者往往不易检出,即使感染晚期因取材局限阳性率也只有50%左右,且不易被患者接受。在旋毛虫感染的过程中,幼虫的排泄分泌(excretory-secretory, ES)蛋白主要来自于杆状体分泌颗粒,直接暴露于宿主的免疫系统,是诱导宿主产生免疫应答的主要靶抗原,可刺激宿主产生强烈的免疫反应,被国际旋毛虫病委员会(International Conference on Trichinellosis, ICT)推荐用于ELISA或Western blotting检测血清中的旋毛虫抗体,但在旋毛虫感染早期抗体检出率较低,且与其他寄生虫感染者之间存在一定的交叉反应。本研究对旋毛虫肌幼虫ES蛋白进行双向电泳(two dimensional gel electrophoresis,2-DE)、 Western blotting和质谱分析,并对筛选出的旋毛虫保护性抗体靶向抗原(antigen targeted by protective antibodies, ATPA, GenBank No. gi|404638)基因进行了克隆、表达及免疫学鉴定,并将其用于旋毛虫实验感染小鼠和旋毛虫病人血清特异性抗体的检测,对寻找旋毛虫病免疫诊断候选抗原及研制高保护性旋毛虫病疫苗具有一定的科学意义。材料与方法1旋毛虫虫种、血清与实验动物本文所用虫种为河南省南阳猪源旋毛虫(T1)由本实验室传代保种。检测血清包括乡土旋毛虫(T2)、布氏旋毛虫(T3)、伪旋毛虫(T4)、纳氏旋毛虫(T7)、裂头蚴、弓形虫与日本血吸虫感染小鼠血清,以及旋毛虫病人与其他寄生虫病人血清。实验动物为昆明小鼠和雌性BALB/c小鼠。2应用免疫蛋白组学筛选旋毛虫肌幼虫排泄分泌蛋白中的早期诊断抗原通过人工消化法和改良贝氏法收集纯净的旋毛虫肌幼虫,于体外培养后获得其ES蛋白。将旋毛虫肌幼虫经灌胃法感染BALB/c小鼠(300条/只),在感染后14～42天隔天进行尾静脉采血,分离血清,应用肌幼虫ES蛋白分别通过ELISA和Western blotting对感染小鼠血清中的旋毛虫抗体进行检测,将感染后最早出现旋毛虫抗体的血清用于后续实验。将旋毛虫肌幼虫ES蛋白进行2-DE后转印至PVDF膜,通过Western blotting应用旋毛虫感染早期抗体阳性血清对旋毛虫肌幼虫ES蛋白进行识别,将阳性反应的蛋白点进行质谱鉴定。3旋毛虫ATPA基因的克隆、表达及鉴定将旋毛虫肌幼虫ES蛋白中分子量为30～40kDa、通过质谱被成功鉴定的阳性反应蛋白点在2-DE凝胶和胶片上的灰度值及其比值分别进行比较,从中选出比值最大的ATPA作为研究对象,应用基因工程技术对旋毛虫ATPA基因进行分子克隆、表达和纯化,将纯化后的rATPA蛋白免疫BALB/c小鼠获得其免疫血清,通过Western blotting分析其抗原性和免疫原性,应用RT-PCR观察ATPA基因在旋毛虫不同发育期(成虫、新生幼虫、成囊前期幼虫及成囊期幼虫)是否转录；应用间接免疫荧光抗体试验(IFT)对ATPA蛋白在旋毛虫不同发育期的表达及其在虫体组织的定位进行分析。4rATPA用于旋毛虫感染小鼠与旋毛虫病人血清抗体的检测应用rATPA蛋白与肌幼虫ES蛋白分别建立检测旋毛虫抗体IgG的rATPA-ELISA与ES-ELISA方法,分别用于检测旋毛虫T2、T3、T4和T7)感染小鼠及其他寄生虫感染小鼠、旋毛虫病人及其他寄生虫病人血清抗体观察rATPA-ELISA诊断旋毛虫病的敏感性与特异性。将30只雌性6周龄BALB/c小鼠随机分为重度(500条/只)、中度(300条/只)、轻度(100条/只)3个感染组(每组10只),应用rATPA-ELISA与ES-ELISA同时对3组小鼠感染后2-42天的血清抗体进行检测,观察rATPA-ELISA对旋毛虫感染早期及轻度感染的诊断价值。5统计学处理采用SPSS17.0统计分析软件,采用卡方检验和重复资料的方差分析进行数据处理和统计分析,检验水平为α=0.05。结果1应用免疫蛋白组学筛选旋毛虫肌幼虫排泄分泌蛋白中的早期诊断抗原应用ELISA及Western blotting方法在小鼠感染旋毛虫后18天可检测出血清抗旋毛虫抗体。分别将200μg和800μg旋毛虫肌幼虫ES蛋白经10%和12%凝胶进行2-DE,可将分子量为40～60kDa和30～40kDa的蛋白进行很好的分离,分别检测到约33个和150个蛋白点,转印至PVDF膜后应用旋毛虫感染后18天的小鼠血清进行Western blotting分析,发现有31个阳性反应蛋白点,分别经胰酶消化后进行MALDI-TOF/TOF-MS分析,共鉴定出7种旋毛虫蛋白,分别为：2种丝氨酸蛋白酶[serine proteases, SP1.2(gi|168805931)和SP1.3(gi|13641204, gi|168805933)]、DNase II (gi|339241449, gi|316974621)、2种假定的胰蛋白酶(putative trypsin, gi|339241891和gi|339241897)、保护性抗体靶向抗原(ATPA, gi|404638)及保守的假定蛋白(conserved hypothetical protein, gi|316966524)。对成功鉴定的已知功能的7种旋毛虫蛋白进行功能分类,7种旋毛虫蛋白均具有催化和水解活性,且与代谢过程有关。2ATPA基因的克隆、表达及鉴定通过生物信息学软件对ATPA基因进行预测,发现其不含跨膜区,但含有信号肽序列(切割位点位于17～18位氨基酸残基间),且具有良好的抗原性。通过RT-PCR获得ATPA基因,并将其连接至表达载体pMAL-c2X,成功构建了ATPA基因的重组表达质粒pMAL-c2X-ATPA。对重组质粒pMAL-c2X-ATPA进行IPTG诱导表达,SDS-PAGE分析发现在74kDa处出现一条明显的蛋白带(载体蛋白43kDa+目的蛋白31kDa),表明重组蛋白表达成功,可溶性分析发现重组蛋白以可溶与包涵体2种形式表达。应用Amylose树脂预装柱对表达的rATPA进行纯化,将纯化后的rATPA蛋白免疫BALB/c小鼠获得免疫血清,ELISA检测rATPA免疫血清的效价为1：106。Western blotting结果显示,rATPA可被旋毛虫感染小鼠血清及rATPA免疫血清识别,rATPA免疫血清均可识别旋毛虫肌幼虫可溶性蛋白和ES蛋白的天然ATPA。RT-PCR分析发现,ATPA基因在旋毛虫4个发育期(成虫、新生幼虫、成囊前期幼虫和成囊期幼虫)均有转录；IFT结果显示,ATPA蛋白在以上4个发育期均有表达,且主要定位于虫体表皮与杆状体部位。3rATPA用于旋毛虫感染小鼠血清抗体的检测应用rATPA-ELISA检测T1、T2、T3、T4和T7感染小鼠的血清抗体阳性率分别为96.67%(29/30)、90.00%(27/30)、93.33%(14/15)、40.91%(9/22)和93.75%(15/16),应用ES-ELISA法检测T1、T2、T3、T4和T7感染小鼠的血清抗体,分别为100%(30/30)、100%(30/30)、100%(15/15)、90.91%(20/22)和100%(16/16)；统计学分析表明rATPA-ELISA与ES-ELISA检测T1、T2、T3和T7感染小鼠血清抗体阳性率的差异无统计学意义(P>0.05),但在检测T4感染小鼠血清时,rATPA-ELISA的阳性率低于ES-ELISA (P<0.05)。应用rATPA-ELISA与ES-ELISA检测旋毛形线虫感染小鼠血清的抗体阳性率分别为96.67%(29/30)和100%(30/30)(P>0.05),检测裂头蚴感染小鼠血清的抗体阳性率分别为16.13%(5/31)和3.22%(1/31)(P>0.05),检测日本血吸虫感染小鼠血清的抗体阳性率分别为68.75%(11/16)和6.25%(1/16)(P<0.05)。rATPA-ELISA与ES-ELISA检测弓形虫感染小鼠血清及正常小鼠血清均为阴性。4rATPA-ELISA检测不同剂量旋毛虫感染小鼠后不同时间的血清抗体水平在重、中、轻度旋毛虫感染小鼠,rATPA-ELISA首次检测到旋毛虫抗体的时间分别是感染后8、12及14天,ES-ELISA首次检测到旋毛虫抗体的时间分别是感染后10、8及10天。在旋毛虫重度感染组,感染后12天和14天,rATPA-ELISA检测的抗体阳性率分别为20%和40%,ES-ELISA检测的抗体阳性率分别为80%和100%(P<0.05)；在旋毛虫中度感染组,感染后10、12和14天,rATPA-ELISA检测的抗体阳性率分别为0、20%和30%,ES-ELISA检测的抗体阳性率分别为70%、100%和100%(P<0.05)；在旋毛虫轻度感染组,感染后12～24天隔天(感染后12、14、16、18、20、22和24天),rATPA-ELISA检测的抗体阳性率分别为0、10%、10%、10%、30%、30%和30%,ES-ELISA检测的抗体阳性率分别为10%、60%、80%、80%、80%、100%和100%(P<0.05)。在轻、中、重度旋毛虫感染小鼠,rATPA-ELISA检测到旋毛虫抗体100%的时间分别是在感染后30、22及28天,ES-ELISA检测到旋毛虫抗体100%的时间分别为感染后22、12及14天。结果表明,在旋毛虫重、中及轻度感染组,在感染后16、16及26天,rATPA-ELISA与ES-ELISA检测的血清抗体阳性率的差异均无统计学意义(P>0.05)。rATPA-ELISA检测重、中、轻度感染小鼠血清抗体水平的差异无统计学意义(F=2.049,P>0.05),感染后不同时间的抗体水平之间的差异有统计学意义(F=219.924,P<0.05),感染后检测时间与感染剂量之间存在交互关系(F=3.311,P<0.05)；两两比较结果显示,感染后10～16天及24～28天血清抗体水平均呈升高趋势(P<0.05)。ES-ELISA检测重、中、轻度感染小鼠血清抗体水平的差异具有统计学意义(F=5.901,P<0.05),感染后不同时间的抗体水平之间的差异有统计学意义(F=478.276,P<0.05),感染后检测时间和感染剂量之间存在交互关系(F=5.710,P<0.05)；两两比较结果显示感染后8-32天血清抗体水平均呈升高趋势(P<0.05)。5rATPA用于旋毛虫病人血清抗体的检测rATPA-ELISA和ES-ELISA检测旋毛虫病人血清抗体阳性率均为100%(22/22), rATPA-ELISA仅在日本血吸虫病人血清检测到1例抗体阳性(5.000%),而检测并殖吸虫病人、华支睾吸虫病人、棘球蚴病人、猪囊尾蚴病人、裂头蚴病人及健康人血清抗体均为阴性。rATPA-ELISA检测旋毛虫病人血清抗体的敏感性与特异性分别为100%与99.13%。ES-ELISA检测日本血吸虫病人、并殖吸虫病人、华支睾吸虫病人、棘球蚴病人、猪囊尾蚴病人、裂头蚴病人抗体抗体阳性率分别为20.00%(4/20)、5-00%(1/20)、14.29%(1/7)、25.00%(5/20)、25.00%(5/20)和12.50%(1/8),而检测健康人血清抗体为阴性。rATPA-ELISA和ES-ELISA检测日本血吸虫病人、并殖吸虫病人、华支睾吸虫病人和裂头蚴病人血清的差异无统计学意义(P>0.05),而在检测棘球蚴病人和猪囊尾蚴病人血清时,rATPA-ELISA优于ES-ELISA (P<0.05)。结论1.应用2-DE与质谱分析从旋毛虫肌幼虫ES蛋白中鉴定出了7种旋毛虫蛋白(2种丝氨酸蛋白酶、DNase Ⅱ、2种胰蛋白酶、保护性抗体靶向抗原及保守的假定蛋白),均具有催化和水解活性,这7种蛋白对旋毛虫病可能具有潜在的早期诊断价值。2.成功构建了旋毛虫ATPA基因的重组表达质粒pMAL-c2X-ATPA, rATPA以可溶与包涵体2种形式表达。ATPA基因在旋毛虫4个发育期(成虫、新生幼虫、成囊前期幼虫和成囊期幼虫)均有转录与表达,主要定位于虫体表皮与杆状体。3.rATPA-ELISA检测旋毛虫感染小鼠和旋毛虫病人血清抗体具有良好的敏感性与特异性,提示rATPA具有用于旋毛虫病的血清学诊断的潜能。更多还原

【Abstract】 Trichinellosis, caused by the ingestion of raw or inadequately cooked meat containing the infective larvae of the nematode genus Trichinella, is a food-borne parasitic zoonosis with worldwide distribution. The diagnosis of trichinellosis is rather difficult because the signs and symptoms are non-specific. Up to now, a definitive diagnosis of human trichinellosis can be made only by detecting larvae in a muscle biopsy or by highly specific immunodiagnositic tests. The sensitivity of muscle biopsy depends on the amount of muscle sample tested and the degree of infection. Muscle biopsy is not sensitive to the light infections and the early stage of infection. Even during the late stage of infection, the positive rate of muscle biopsy is only50%due to limitations of selecting muscular tissue. Besides, muscle biopsy is difficult to be accepted by patients. During the process of infection by Trichinella, the excretory-secretory (ES) proteins of muscle larvae (ML), mainly come from the stichocyte granules, are directly exposed to the host’s immune system, and can induce a strong immune response involving the generation of specific antibodies as they are easily targeted by the host’s immune system. The ES proteins of T. spiralis ML were recommended to be used in ELISA or Western blotting for detecting anti-Trichinella antibodies by International Commission on Trichinellosis (ICT), but their main disadvantages are the false negative results during the early stage of infection and cross-reaction with other parasites. In this study, the ES proteins from ML of T. spiralis were analysed by two dimensional gel electrophoresis (2-DE), Western blotting combined with mass spectrometry (MS), and the gene of antigen targeted by protective antibodies (ATPA, GenBank No. gi|404638) was selected to be cloned, expressed and anylased by immunological methods. Besides, the recombinant protein was applied to detect the specific anti-Trichinella antibodies in serum from the mice infected with T spiralis and patient’s sera with trichinellosis, and it will lay the foundation for searching the candidates of immunodiagnostic antigens for trichinellosis and the effective protective antigens for trichinellosis vaccine.Materials and methods1Trichinella spp., serum and experimental animalsT. spiralis isolate (T1) used in this study was maintained by serial passages in Kunming mice in our laboratory. Mouse serum samples were infected with Trichinella native (T2), T. britovi (T3), T. pseudospiralis (T4), T. nelsoni (T7), Toxoplasma gondii and Schistosoma japanicum, as well as patient’s sera with trichinellosis and other parasitosis. Experimental animals were female Kunming mice and BALB/c mice.2Identification of early diagnostic antigens from the ES proteins of T. spiralis muscle larvae using immunoproteomicsThe muscle larvae of T. spiralis were collected by artificial digestion and modified Baermann’s methods, and the ES proteins were obtained by culturing the ML in vitro. BALB/c mice were orally infected with300larvae/mouse and the tail vein blood was daily collected from each mouse before infection and during14-42days post-infection (dpi) on alternate days, respectively. Anti-Trichinella IgG antibodies in sera from infected mice at14-42dpi were assayed by ELISA and Western blotting methods using T. spiralis ML ES proteins as antigens. The sera which firstly detected the specific antibodies by the above-mentioned two methods were used in the following experiments. The ES proteins from T. spiralis ML were separated by2-DE, then were transferred onto polyvinylidene difluoride (PVDF) membranes and probed with early mouse sera infected with T. spiralis, and the immunoreactive protein spots were identified and characterized by MS.3Cloning, expression and identification of T. spiralis ATPA geneThe volumes of protein spots successfully identified by MS with30-40kDa from ES proteins of T. spiralis ML in2-DE and Western blotting and the ratios were compared. Out of these proteins, ATPA had the largest ratio and was selected for the further study. The gene of ATPA was cloned, expressed and purified. BALB/c mice were immunized with rATPA, and the immune sera were collected. The antigenicity and immunogenicity of rATPA were identified by Western blotting. RT-PCR was carried out to observe the transcription of ATPA gene in different development stages [adult worms (AD), new-born larvae (NBL), pre-encapsulated larvae (PEL) and ML] of Trichinella. The expression of ATPA protein in different development stage and location of rATPA were observed by IFT.4Application of rATPA for detection of Trichinella-specific antibodies in mouse sera infected with T. spiralis and patient sera with trichinellosisrATPA-ELISA and ES-ELISA were established using rATPA and ES proteins of T. spiralis ML, respectively. They were used to assay anti-Trichinella IgG antibodies in sera from mice infected with different species of Trichinella (T1, T2, T3, T4, and T7) and other parasites, as well as patient’s sera with trichinellosis and other parasitosis, and their sensitivity and specificity were evaluated.30female BALB/c mice were randomly divided into3groups (10mice/group):heavily infected group (500larvae/mouse), moderately infected group (300larvae/mouse), and lightly infected group (100larvae/mouse). The mice of infected group were orally inoculated with of T. spiralis muscle larvae. rATPA-ELISA and ES-ELISA were used to assay anti-Trichinella IgG antibodies in sera from mice infected with T. spiralis on alternate days during2-42dpi, and the value of rATPA-ELISA for diagnosis of trichinellosis during the the early stage of infection and light infections were evaluated. 5Statistical analysisAll statistical analyses of data were done with SPSS for Windows version17.0. Chi-square test and repeated measures of analysis of variance (ANOVA) were used, and the level of significance used was5%(P<0.05).Results1Identification of early diagnostic antigens from the ES proteins of T. spiralis muscle larvae using immunoproteomicsAnti-Trichinella IgG antibodies in sera from infected mice at14-42dpi were assayed by ELISA and Western blotting. The specific antibodies were firstly detected at18dpi by the above-mentioned two methods, and then these sera were used for the following experiments.200μg and800μg ES proteins of T. spiralis ML were separated by2-DE on10%and12%gels, the protein spots with40-60kDa and30-40kDa can be separated very well, and more than33and150spots were detected. After transferred to PVDF membranes, the spots were probed with18dpi mouse sera infected with T. spiralis, and there were31spots displaying reactivity to the infection sera at18dpi. These spots recognized by the infection sera at18dpi were digested by trypsin and then analysed by MALDI-TOF/TOF-MS, and these spots were identified to correlate with7different proteins of T. spiralis, including two serine proteases [serine proteases, SP-1.2(gi|168805931) and SP-1.3(gi|13641204, gi|168805933)], one deoxyribonuclease (DNase) Ⅱ (gi|339241449, gi|316974621), two kinds of trypsin (putative trypsin, gi|339241891and gi|339241897), one antigen targeted by protective antibodies (ATPA, gi|404638) and one conserved hypothetical protein (gi|316966524). The7kinds of proteins of T. spiralis identified were putatively annotated using the GO categories tool. The7proteins of T. spiralis had catalytic and hydrolase activity, and they were also associated with metabolic process.2Cloning, expression and identification of T. spiralis ATPA genePrediction of ATPA by soft wares of bioinformatics showed that the ATPA had no transmembrane domain, had a cleavable signal peptide (from1to17) and with possible cleavage site between17aa and18aa, and had good antigenicity. The ATPA gene obtained by RT-PCR was cloned to pMAL-c2X expression vector, and the recombinant plasmid pMAL-c2X-ATPA was constructed successfully. The recombinant plasmid pMAL-c2X-ATPA was induced by IPTG and the recombinant fusion protein74kDa (tag protein43kDa+ATPA31kDa) could be found by SDS-PAGE. The rATPA was expressed successfully, and existed in forms of both soluble protein and inclusion body in E. coli strain TB1. The desired protein was purified by Amylose pre-packed column after obtaining the supernatant. BALB/c mice were immunized with rATPA, and the immune sera were collected. The specific IgG antibody titer of immune sera were assayed by an indirect enzyme-linked immunosorbent assay (ELISA) using rATPA, and the IgG antibody titer of immune sera against rATPA was1:106. Western blotting showed that the rATPA could be recognized by the mouse sera infected with T. spiralis and immune sera against rATPA, but it was not immunostained with normal serum. And the native ATPA protein in soluble proteins and ES proteins from T. spiralis ML were recognized by immune sera against rATPA. RT-PCR results showed that ATPA mRNA was transcribed in different development stages (AD, NBL, PEL and ML) of Trichinella. The IFT results showed that the ATPA protein was expressed in the4different development stages, and the bright green fluorescence was mainly localized at the cuticle and stichosome of the worms.3Sensitivity and specificity of rATPA-ELISA for detection of Trichinella-specific antibodies in mouse sera infected with T. spiralisPositive rates of the specific enti-Trichinella IgG antibodies from mouse sera infected with Trichinella T1, T2, T3, T4, and T7were96.67%(29/30),90.00%(27/30),93.33%(14/15),40.91%(9/22), and93.75%(15/16) by rATPA-ELISA, while the positive rates of the above sera were100%(30/30),100%(30/30),100%(15/15),90.91%(20/22), and100%(16/16) by ES-ELISA. There was no significant difference in the positive rates of mouse sera infected with Trichinella T1, T2, T3, and T7between rATPA-ELISA and ES-ELISA (P>0.05); but as for the positive rates of mouse sera infected with Trichinella T4, rATPA-ELISA was lower than ES-ELISA (P<0.05).Positive rates of the specific anti-Trichinella IgG antibodies from mouse sera infected with T. spiralis by rATPA-ELISA and ES-ELISA were96.67%(29/30) and100%(30/30)(P>0.05). Positive rates of antibodies from mouse sera infected with plerocercoids of Spirometra mansoni (sparganum) by rATPA-ELISA and ES-ELISA were16.13%(5/31) and3.22%(1/31)(P>0.05). Positive rates of mouse sera infected with S. japanicum by rATPA-ELISA and ES-ELISA were68.75%(11/16) and6.25%(1/16)(P<0.05). However, the mouse sera collected from mice infected with T. gondii and before infection showed negative response by both rATPA-ELISA and ES-ELISA.4Serum IgG levels at different times in the mice experimentally infected with different dose of T. spiralis muscle larvaeIn lightly, moderately and heavily infected group, the specific anti-Trichinella IgG antibodies were firstly detected at14dpi,12dpi, and8dpi by rATPA-ELISA, while the antibodies were firstly detected at10dpi,8dpi, and10dpi by ES-ELISA, respectively. In heavily infected group, the positive rates of the specific anti-Trichinella IgG antibodies on12dpi and14dpi by rATPA-ELISA were20%and40%, while the positive rates of antibodies by ES-ELISA were80%and100%(P <0.05). In mouse sera of moderately infected group, the positive rates of the specific anti-Trichinella IgG antibodies on10dpi,12dpi, and14dpi by rATPA-ELISA were0,20%, and30%, while the positive rates of antibodies by ES-ELISA were70%,100%, and100%(P<0.05). In mouse sera of lightly infected group, the positive rates of the specific anti-Trichinella IgG antibodies on alternate days during12-24dpi by rATPA-ELISA were0,10%,10%,10%,30%,30%, and30%, while the positive rates of antibodies by ES-ELISA were10%,60%,80%,80%,80%,100%, and100%(P<0.05). The specific anti-Trichinella IgG antibodies were detected100%at30dpi,22dpi and28dpi a in lightly, moderately and heavily infected group by rATPA-ELISA, while the antibodies were detected100%at22dpi,12dpi and14dpi by ES-ELISA. There was no significant difference in the positive rates of mouse sera in lightly, moderately and heavily infected group during26dpi,16dpi and16dpi between rATPA-ELISA and ES-ELISA (P>0.05).There was no significant difference on the specific anti-Trichinella IgG antibodies level in heavily, moderately and lightly infected group by rATPA-ELISA (F=2.049,P>0.05); the difference of serum antibody levels at different times by rATPA-ELISA was statistically significant (F=219.924, P<0.05); there was interaction between time and the dose of inoculation (F=3.311,P<0.05). The results of paired comparison showed that the serum antibody level was raised during10-16dpi and24-28dpi (P<0.05). There was significant difference on the specific anti-Trichinella IgG antibodies level in heavily, moderately and lightly infected group by ES-ELISA (F=5.901, P<0.05); the difference of serum antibody levels at different times by ES-ELISA was statistically significant (F=478.276, P<0.05); there was interaction between time and the dose of inoculation (F=5.710, P<0.05). The results of paired comparison showed that the serum antibody level was raised during8-32dpi (P<0.05).5Sensitivity and specificity of rATPA-ELISA for detection of Trichinella-specific antibodies in patient’s sera with trichinellosisThe sensitivity of both rATPA-ELISA and ES-ELISA in detecting the serum samples of patients with trichinellosis was100%(22/22). Using rATPA-ELISA, only one case of patient’s sera with schistosomiasis was positive, while no positive reaction was detected in patients’sera with paragonimiosis, clonorchiosis, echinococcosis, cysticercosis and sparganosis, as well as healthy persons’ sera. The sensitivity and specificity of rATPA-ELISA for detecting specific anti-Trichinella antibodies in sera of patients with trichinellosis were100%and99.13%, respectively. Using ES-ELISA, Positive rates of the specific anti-Trichinella IgG antibodies from patients’ sera with schistosomiasis, paragonimiosis, clonorchiosis, echinococcosis, cysticercosis and sparganosis were20.00%(4/20)、5.00%(1/20、14.29%(1/7)、25.00%(5/20)、25.00%(5/20)和12.50%(1/8), but no positive reaction was detected in healthy persons’ sera. There was no significant difference in the detection of patients’ sera with schistosomiasis, paragonimiosis, clonorchiosis, and sparganosis (P>0.05) between rATPA-ELISA and ES-ELISA; but as for detection of patients’ sera with echinococcosis and cysticercosis, rATPA-ELISA was better than ES-ELISA (P <0.05).Conclusions1. Seven proteins of T. spiralis (two serine proteases, one DNase II, two kinds of trypsin, one antigen targeted by protective antibodies and one conserved hypothetical protein) were identified from muscle larval excretory-secretory proteins. All of the7proteins of T. spiralis had catalytic and hydrolase activity, and might have potential values of early diagnosis for trichinellosis.2. The recombinant plasmid pMAL-c2X-ATPA was constructed successfully, and the rATPA existed in forms of both soluble protein and inclusion body. The ATPA mRNA was transcribed in different development stages (AD, NBL, PEL and ML), the protein of ATPA was expressed in the4different development stages, and was mainly localized at the cuticle and stichosome of the worms.3. rATPA-ELISA which was used to detect the specific anti-Trichinella IgG antibodies in mouse sera infected with T. spiralis and patient sera with trichinellosis, had good sensitivity and specificity, and rATPA could be potential for serological diagnosis for trichinellosis.更多还原