细粒棘球绦虫重组双歧杆菌-Eg95疫苗诱导小鼠抗感染的保护性免疫作用

Anti-infective immunity of recombinant Bifutobacteria bifMum-Eg95 vaccine of Echinococcus granulosusin mice

目的探讨细粒棘球绦虫（Eg）重组双歧杆菌（Bb）-Eg95疫苗免疫小鼠后对Eg原头节攻击感染的保护性免疫作用。方法雌性BALB／c小鼠56只，12～14周龄，体质量20～25g。将重组Bb—Eg95疫苗分别采用皮下注射、肌肉注射、鼻腔黏膜接种和口服灌胃4种途径免疫BALB／c小鼠，同时设有空载体、Bb和液体培养基（MRS）对照组，每组8只。免疫后8周，用Eg原头节攻击感染，感染后25周剖杀小鼠，分离细粒棘球蚴包囊并称重，计算减蚴率；取血，分离血清，常规酶联免疫吸附测定法（ELISA）测定血清免疫球蛋白IgG及其亚类和kE水平；取脾，分离脾细胞，采用细胞培养的方法，在脾细胞悬液中分别加入Eg粗抗原（EgAg）和刀豆素A（ConA），四甲基偶氮唑盐（MTT）比色法检测脾淋巴细胞增殖反应。组间比较采用方差分析，进一步两两比较采用LSD-t检验。结果小鼠体内细粒棘球蚴质量组间比较差异有统计学意义（F=11．062，P〈0．05）。其中皮下注射组、肌肉注射组、鼻腔接种组和口服灌胃组细粒棘球蚴质量[（0．050±0．013）、（0．050±0．019）、（0．028±0．016）、（0．031±0．01）g]明显低于MRS对照组[（O．075±0．019）g，P均〈0．01]，鼻腔接种组和口眼灌胃组细粒棘球蚴质量明显低于皮下注射组和肌肉注射组（P均〈0．05）；减蚴率的高低与小鼠体内绑粒棘球蚴质量变化成反比。小鼠血清IgG、IgG2a、IgG2b、IgGl、IgG3、IgE水平（吸光度，A），组间比较差异有统计学意义（F=21．774、36．977、27．071、14．746、10．131、9．444，P〈0．05或’P〈0．01）。其中血清IgG、IgG2a和IgG2b水平皮下注射组（0．022±0．004、0．007±0．002、0．008±0．002）、肌肉注射组（O．023±0．003、0．008±0．002、0．007±0．002）、鼻腔接种组（0．032±0．007、0．012±0．002、0013±0．004，）和口服灌胃组（0．028±0．006、0．010±0．003、0．010±0．002）均显著高于NRS对照组（O．015±0．002、0．002±0．001、0.003±0．001，P均〈0．01），鼻腔接种组和1：3服灌胃组高于皮下注射组和肌肉注射组（P〈0．05或P〈0．01）；小鼠血清IgGl、IgG3和IgE水平，皮下注射组（0．004±0．001、0．003±0．002、0．004±0．002）、肌肉注射组（0．004±0．001、0．004±0．001、0．004±0．002）、鼻腔接种组（0．005±0．002、0．005±0．003、0．005±0．002）和口服灌胃组（0．005±0．001、0．004±0．002、0．004±0．003）均显著低于MRS对照组（0．009±0．001、0．009±0．002、0．009±0．001，P均〈0．01）。小鼠脾淋巴细胞增殖水平在脾细胞悬液、脾细胞悬液＋EgAg、脾细胞悬液＋ConA时，组间比较差异有统计学意义（F=63．975、359．833、167．399，P均〈0．01），组内组间比较差异有统计学意义（F=6741．955、4953．667、869．320、201．235、175．413、139．653、169．994，P均〈O．01），其中在加入EgAg或ConA时，脾淋巴细胞增殖水平明显高于单纯脾细胞悬液，而且加入ConA高于EgAg（P均〈0．01）。结论细粒棘球绦虫重组Bb-Eg95疫苗免疫小鼠，能有效的诱导小鼠产生保护性的免疫作用。

Objective To investigate the protective immunity in mice immunized With recombinant Bifidobacteria bifidum（Bb）-Eg95 vaccine of Echinococcus granulosus （Eg）.and challenged with Eg protoseoleces.Methods Fifty-six female BALB/c mice 12 - 14 weeks old and weighed 20 - 25 g were vaccinated with the recombinant Bb-Eg95 vaccine subcutaneously, intramuscularly, intranasally and orally, respectively, with blank vector, Bb and medium of solution（MRS） as control, 8 mice in each group. Mice were challenged with Eg protoscoleces on week 8 after immunization and killed on week 25 after infection. The weight of hydatid cyst was measured and the decreased larva rate was calculated. Sera were collected to determine the levels of IgE, IgG and its subclasses by enzyme linked immunosorbent assay（ELISA）. Splenocytes were collected and cultivated to test the proliferation of splenocytes using methyhetrazolium （MTr） assay under EgAg and concanavalin A （ConA） stimulation. The results were compared with analysis of variance and the comparison between two groups was performed with LSD-t test. Results There was significant difference in the weight of hydatid cyst between groups（F = 1t.062, P 〈 0.05）. Compared with MRS control group[ （0.075 ± 0.019）g], the hydatid cyst weight decreased in subcutaneous group [ （0.050 ± 0.013）g], intramuscular group[ （0.050 ± 0.019）g], intranasal group[ （0.028 ± 0.016）g] and oral group [ （0.031 ± 0.018）g, all P 〈 0.01 ）. Compared with subcutaneous and intramuscular groups, the hydatid cyst weight decreased in intranasal and oral groups（all P 〈 0.05）. The decreased larva rate was inversely proportional to the weight of hydatid cyst. There was significant difference in the levels（obsorbancy, A ） of IgG, IgG2a, IgG2b, IgG1, IgG3 and IgE between these groups（F = 21.774, 36.977, 27.071, 14.746, 10.131, 9.444, P 〈 0.05 or P 〈 0.01 ）. Compared with MRS control group（0.015 ± 0.002, 0.002 ± 0.001, 0.003 ± 0.001）, the levels of IgG, IgG2a and IgG2b increased in subcutaneous group（0.022 ± 0.004, 0.007 ± 0.002, 0.008 ± 0.002）, intramuscular group （0.023 ± 0.003, 0.008 ± 0.002, 0.007 ± 0.002）, intranasal group（0.032 ± 0.007, 0.012 ± 0.002, 0.013 ± 0.004） and oral group（0.028 ± 0.006, 0.010 ± 0.003, 0.010 ± 0.002, P 〈 0.05 or P 〈 0.01）. Compared with subcutaneous and intramuscular .groups, the levels of IgG, IgG2a and IgG2b increased in intranasal and oral groups（P 〈 0.05 or P 〈 0.01 ）. Compared with MRS control group（0.009 ± 0.001,0.009 ± 0.002, 0.009 ± 0.001）, the levels of IgG1, IgG3 and IgE decreased in subcutaneous group（0.022 ± 0.004, 0.007 ± 0.002, 0.008 ± 0.002）, intramuscular group（0.004 ± 0.001, 0.004 ± 0.001, 0.004 ± 0.002）, intranasal group（0.005 ± 0.002, 0.005 ± 0.003, 0.005 ± 0.002） and oral group（0.005 ± 0.001, 0.004 ± 0.002, 0.004 ± 0.003, all P〈 0.01）. There was significant difference in the proliferation of splenocytes in the supernatant of cultured splenoeyte, of cultured splenocyte EgAg and of cultured splenocyte ＋ ConA（F = 63.975, 359.833, 167.399, P 〈 0.01 ）. There was significant difference in the proliferation of splenocytes inside groups（F = 6741.955, 4953.667, 869.320, 201.235, 175.413, 139.653, 169.994, all P 〈 0.01 ）. Compared with the cultured splenocyte the proliferation of splenocytes increased in the cultured splenocyte ＋ EgAg and splenocyte ＋ ConA （all P 〈 0.01 ）. Compared with the cultured splenocyte ＋ EgAg, the proliferation of splenocytes increased in the cultured splenocyte ＋ ConA（P 〈 0.01 ）. Conclusion An effective and protective immunity is induced by the recombinant Bb-Eg95 vaccine of Eg in mice.