豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗誘導(dǎo)仔豬免疫應(yīng)答的動(dòng)態(tài)觀察

王靈軍，劉美辰，周　泠，楊鳳嬌，賈　啟，江　楠，周必英

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豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗誘導(dǎo)仔豬免疫應(yīng)答的動(dòng)態(tài)觀察

王靈軍1，劉美辰1，周泠2，楊鳳嬌1，賈啟1，江楠1，周必英1

1.遵義醫(yī)學(xué)院寄生蟲(chóng)學(xué)教研室，遵義563000；2.遵義醫(yī)學(xué)院附屬醫(yī)院中醫(yī)肛腸科，遵義563000

摘要：目的研究豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗免疫仔豬后誘導(dǎo)免疫應(yīng)答的動(dòng)態(tài)變化。方法將16頭40 d齡健康仔豬均分為4組：重組Bb-TSO45W-4B-TSOL18疫苗組、重組Bb-TSO45W-4B疫苗組、重組Bb-TSOL18疫苗組和雙歧桿菌液體培養(yǎng)基(MRS)對(duì)照組。各疫苗組均以1011CFU灌胃免疫，間隔2周免疫1次，共免疫2次。在免疫后0、2、4、6、8周采集仔豬前腔靜脈血，分離血清和外周血淋巴細(xì)胞(PBMC)。采用ELISA法檢測(cè)仔豬血清IgG、IgG1及IgG2a水平和PBMC培養(yǎng)上清液IL-2、IFN-γ、IL-4及IL-10水平；MTT比色法檢測(cè)PBMC增殖水平；FCM檢測(cè)CD4+和CD8+T細(xì)胞亞群。結(jié)果各疫苗組血清IgG、IgG1、IgG2a水平均在免疫后2～8周升高，均在免疫后4周、6周、4周達(dá)較高水平；PBMC增殖水平均在免疫后2～8周升高，均在免疫后6周達(dá)較高水平；CD4+和CD8+T細(xì)胞水平均在免疫后2～8周升高，均在免疫后8周和6周達(dá)較高水平；PBMC培養(yǎng)上清液IL-2、IFN-γ、IL-4、IL-10水平分別在免疫后2～6周、2～8周、4～6周和2～8周升高，分別在免疫后4周、6周、4周和8周達(dá)較高水平。各疫苗組上述指標(biāo)與MRS對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；重組Bb-TSO45W-4B-TSOL18疫苗組與重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗免疫仔豬可誘導(dǎo)產(chǎn)生特異性的免疫應(yīng)答，且TSO45W-4B-TSOL18融合抗原疫苗組優(yōu)于TSO45W-4B或TSOL18單一抗原疫苗組。

關(guān)鍵詞：豬帶絳蟲(chóng)；重組Bb-TSO45W-4B-TSOL18疫苗；免疫應(yīng)答；仔豬

Dynamic observation of immune responses induced in piglets

Supported by the National Natural Science Foundation of China (No. 81160206)

豬囊尾蚴病(Cysticercosis)俗稱囊蟲(chóng)病，是由豬帶絳蟲(chóng)(Taeniasolium)幼蟲(chóng)-囊尾蚴引起的一種危害嚴(yán)重的人獸共患寄生蟲(chóng)病。藥物及手術(shù)治療都有其局限性，有保護(hù)作用的疫苗是控制該病流行的有效手段[1]。TSO45W-4B和TSOL18是豬帶絳蟲(chóng)六鉤蚴階段的重要候選疫苗抗原[2]。雙歧桿菌(Bifidobacterium,Bb)作為載體傳遞系統(tǒng)，已在細(xì)菌、病毒、腫瘤、寄生蟲(chóng)等領(lǐng)域得到了廣泛應(yīng)用[3]。本研究擬在成功構(gòu)建豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗[4-5]的基礎(chǔ)上，以該疫苗免疫仔豬，動(dòng)態(tài)觀察其誘導(dǎo)的免疫應(yīng)答。

1材料與方法

1.1疫苗來(lái)源豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗、重組Bb-TSO45W-4B疫苗、重組Bb-TSOL18疫苗由本室構(gòu)建保存[4,6-7]。

1.2實(shí)驗(yàn)動(dòng)物40 d齡健康仔豬16頭，每頭體重約15 kg，購(gòu)自遵義縣三盆鎮(zhèn)養(yǎng)殖場(chǎng)，由遵義醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心飼養(yǎng)管理。1.3主要試劑與儀器純化的豬帶絳蟲(chóng)TSO45W-4B-TSOL18/TSO45W-4B/TSOL18重組抗原由本室制備保存[8-10]；IgG、IgG1和IgG2a試劑盒(美國(guó)Southern Biotech公司)；IL-2、IFN-γ、IL-4和IL-10試劑盒[美國(guó)rndsystem(RD)北京永輝生物技術(shù)有限公司]；異硫氰酸熒光素(FITC)標(biāo)記CD4+單抗和藻紅蛋白(PE)標(biāo)記CD8+單抗(美國(guó)BD Biosciences公司)；ConA(美國(guó)Sigma公司)；RPMI1640培養(yǎng)基、FBS、MTT、DMSO(上海生工生物技術(shù)有限公司)；豬PBMC分離液(大連寶生物公司)。酶標(biāo)儀(山東高密彩虹分析儀器有限公司)；流式細(xì)胞儀由遵義醫(yī)學(xué)院免疫學(xué)教研室提供；37 ℃恒溫水浴箱(上海市躍進(jìn)農(nóng)場(chǎng)醫(yī)療器械廠)。

1.4動(dòng)物分組與免疫將16頭健康仔豬均分為4組：重組Bb-TSO45W-4B-TSOL18疫苗組、重組Bb-TSO45W-4B疫苗組、重組Bb-TSOL18疫苗組和MRS對(duì)照組。疫苗均溶于50 mL MRS，均以1011CFU灌胃免疫，間隔2周免疫1次，共免疫2次。1.5血清收集及抗體檢測(cè)在免疫后0、2、4、6、8周取前腔靜脈血，4 ℃靜置12 h，2 000 r/min離心10 min，收集上清，-20 ℃凍存，采用ELISA法檢測(cè)血清IgG、IgG1和IgG2a水平。

1.6PBMC制備在免疫后0、2、4、6、8周取前腔靜脈血1～2 mL于無(wú)菌EDTA抗凝管，用10% FBS的RPMI1640培養(yǎng)液稀釋2倍。按2∶1緩慢加入到PBMC分離液上，2 500 r/min離心20 min，吸取上下液體交界處的白膜層。用RPMI1640培養(yǎng)液稀釋5倍，洗滌2次，即為淋巴細(xì)胞，細(xì)胞沉淀用RPMI1640培養(yǎng)液懸浮后，用臺(tái)盼藍(lán)檢測(cè)細(xì)胞活力和計(jì)數(shù)，計(jì)數(shù)后，用RPMI1640培養(yǎng)液調(diào)至為5×106/mL。

1.7PBMC增殖反應(yīng)取出細(xì)胞培養(yǎng)板，分別加入1 mL原液(即5×106/mL PBMC)、1 mL原液+10 μL抗原(1 μg/μL)、1 mL原液+10 μL ConA(1 μg/μL)。于37 ℃ 5%CO2培養(yǎng)44 h，加入10 μL MTT(5 mg/mL)，繼續(xù)培養(yǎng)4 h，控干，加入100 μL DMSO，吹打溶解，立即用酶標(biāo)儀測(cè)定OD630nm值。

1.8T細(xì)胞亞群檢測(cè)吸取500 μL 5×106/mL PBMC于1.5 mL EP管，分別加入4 μL CD4+單抗和10 μL CD8+單抗，室溫避光15～30 min，加入1 mL PBS緩沖液，1 200 r/min離心5 min，重復(fù)1次，棄上清，PBS懸浮，用流式細(xì)胞儀檢測(cè)PBMC中CD4+和CD8+T細(xì)胞亞群百分率。

2結(jié)果

2.1血清IgG、IgG1、IgG2a水平重組Bb-TSO45W-4B-TSOL18疫苗組、重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組血清IgG、IgG1、IgG2a水平均在免疫后2～8周升高，分別在免疫后4、6、4周達(dá)較高水平，與MRS對(duì)照組比較，差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)。重組Bb-TSO45W-4B-TSOL18疫苗組高于重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組(P<0.05)。重組Bb-TSO45W-4B疫苗組與重組Bb-TSOL18疫苗組比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，表1-3。

GroupWeek02468rBb-TSO45W-4B-TSOL18vac-cinegroup207.32±0.79209.26±2.71ab272.16±3.36ab252.85±3.76ab237.67±0.54abrBb-TSO45W-4Bvaccinegroup203.74±1.63208.15±2.01a263.58±2.42a245.05±3.66a232.14±1.60arBb-TSOL18vaccinegroup202.85±1.10207.43±2.06a254.44±3.32a243.98±1.36a225.46±3.37aMRScontrolgroup206.26±1.79207.21±3.12209.70±4.18206.58±1.97207.74±2.24

GroupWeek02468rBb-TSO45W-4B-TSOL18vac-cinegroup22.46±0.3324.53±0.26ab25.61±0.26ab27.74±0.13ab24.73±0.06abrBb-TSO45W-4Bvaccinegroup21.79±0.2923.55±0.18a24.57±0.42a25.45±0.16a23.44±0.11arBb-TSOL18vaccinegroup22.58±0.3223.58±0.27a24.43±0.35a25.58±0.28a22.76±1.62aMRScontrolgroup22.21±0.6421.73±1.2723.99±0.6922.47±0.5422.00±0.78

GroupWeek02468rBb-TSO45W-4B-TSOL18vac-cinegroup53.62±0.8089.65±1.91ab94.15±2.85ab73.99±0.30ab65.71±1.33abrBb-TSO45W-4Bvaccinegroup53.47±1.0781.22±0.63a84.17±0.92a69.82±0.98a62.55±0.15arBb-TSOL18vaccinegroup53.25±0.8079.28±0.77a82.14±1.02a69.82±0.45a62.43±0.83aMRScontrolgroup52.98±0.6253.64±0.8352.32±1.5653.30±1.2352.86±0.66

注：與MRS對(duì)照組比較，aP<0.05；與重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組比較，bP<0.05。

Note: Compared with the MRS control group,aP<0.05; Compared with the rBb-TSO45W-4B vaccine group and rBb-TSOL18 vaccine group,bP<0.05.

2.2PBMC增殖水平不加刺激物時(shí)，重組Bb-TSO45W-4B-TSOL18疫苗組、重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組PBMC增殖水平均在免疫后2～8周升高，均在免疫后6周達(dá)較高水平，與MRS對(duì)照組比較，差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)。重組Bb-TSO45W-4B-TSOL18疫苗組高于重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組(P<0.05)。重組Bb-TSO45W-4B疫苗組與重組Bb-TSOL18疫苗組比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。用Ag或ConA刺激時(shí)，PBMC增殖水平類似于不加刺激物結(jié)果，見(jiàn)表4。

GroupStimulantionWeek02468rBb-TSO45W-4B-TSOL18Supernatant0.134±0.0320.366±0.044ab0.535±0.030ab1.367±0.009ab0.742±0.021abvaccinegroupAntigen0.218±0.0070.629±0.016ab0.829±0.039ab1.648±0.021ab0.680±0.018abConA0.567±0.0120.866±0.022ab1.361±0.029ab1.838±0.026ab1.151±0.036abrBb-TSO45W-4BSupernatant0.137±0.0400.332±0.033a0.567±0.025a0.857±0.037a0.550±0.036avaccinegroupAntigen0.241±0.0430.449±0.029a0.661±0.024a0.957±0.038a0.754±0.039aConA0.553±0.0370.645±0.035a0.937±0.012a1.145±0.039a0.824±0.030arBb-TSOL18vaccinegroupSupernatant0.161±0.0330.357±0.022a0.521±0.013a0.871±0.024a0.541±0.040aAntigen0.247±0.0400.444±0.012a0.663±0.026a0.943±0.029a0.737±0.034aConA0.537±0.0200.673±0.010a0.972±0.035a1.130±0.029a0.840±0.035aMRScontrolgroupSupernatant0.114±0.0370.233±0.0240.264±0.0400.239±0.0320.137±0.017Antigen0.274±0.0150.245±0.0330.356±0.0350.254±0.0210.357±0.029ConA0.468±0.0440.439±0.0320.462±0.0170.357±0.0230.452±0.035

注：與MRS對(duì)照組比較，aP<0.05；與重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組比較，bP<0.05。

Note: Compared with MRS control group,aP<0.05; Compared with rBb-TSO45W-4B vaccine group and rBb-TSOL18 vaccine group,bP<0.05.

2.3CD4+和CD8+T細(xì)胞亞群重組Bb-TSO45W-4B-TSOL18疫苗組、重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組CD4+、CD8+T細(xì)胞水平均在免疫后2～8周升高，均在免疫后8周、6周達(dá)較高水平，與MRS對(duì)照組比較，差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)。重組Bb-TSO45W-4B-TSOL18疫苗組高于重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組(P<0.05)。重組Bb-TSO45W-4B疫苗組與重組Bb-TSOL18疫苗組比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，見(jiàn)表5、6。

GroupWeek02468rBb-TSO45W-4B-TSOL18vac-cinegroup48.30±0.7051.98±1.18ab61.43±1.76ab73.33±0.39ab84.50±2.00abrBb-TSO45W-4Bvaccinegroup42.63±2.1247.10±1.20a54.60±1.68a65.95±3.33a74.70±3.48arBb-TSOL18vaccinegroup41.90±1.7547.13±0.87a52.25±1.53a65.50±1.31a75.13±1.28aMRScontrolgroup59.00±4.2552.58±5.4753.15±4.8052.90±7.1565.20±2.28

GroupWeek02468rBb-TSO45W-4B-TSOL18vaccinegroup22.88±1.1267.06±1.20ab74.70±0.74ab81.93±1.59ab78.33±1.20abrBb-TSO45W-4Bvaccinegroup22.33±1.1161.65±1.20a66.30±1.21a73.45±1.18a71.63±1.37arBb-TSOL18vaccinegroup22.05±1.3661.55±1.17a65.60±1.20a72.85±1.84a72.10±1.64aMRScontrolgroup42.88±6.1753.50±6.5738.93±10.7458.58±1.9661.73±5.90

注：與MRS對(duì)照組比較，aP<0.05；與重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組比較，bP<0.05。

Note: Compared with MRS control group,aP<0.05; Compared with rBb-TSO45W-4B vaccine group and rBb-TSOL18 vaccine group,bP<0.05.

2.4IL-2、IFN-γ、IL-4及IL-10水平不加刺激物時(shí)，重組Bb-TSO45W-4B-TSOL18疫苗組、重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組PBMC培養(yǎng)上清液IL-2、IFN-γ、IL-4及IL-10水平分別在免疫后2～6周、2～8周、4～6周和2～8周升高，分別在免疫后4周、6周、4周和8周達(dá)到較高水平，與MRS對(duì)照組比較，差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)。重組Bb-TSO45W-4B-TSOL18疫苗組高于重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組(P<0.05)。重組Bb-TSO45W-4B疫苗組與重組Bb-TSOL18疫苗組比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。用Ag或ConA刺激時(shí)，PBMC培養(yǎng)上清液IL-2、IFN-γ、IL-4及IL-10水平類似于不加刺激物結(jié)果，見(jiàn)表7-10。

GroupStimulantionWeek02468rBb-TSO45W-4B-TSOL18Supernatant152.44±0.87183.69±1.07ab203.53±0.05ab177.54±0.87ab163.05±0.23vaccinegroupAntigen162.51±2.23190.44±1.94ab207.79±1.00ab182.37±0.72ab162.55±0.09ConA165.02±0.82204.16±2.63ab214.74±1.65ab184.51±0.29ab164.67±0.22rBb-TSO45W-4BSupernatant152.93±1.11173.18±1.84a192.55±1.34a171.99±1.69a156.17±2.50vaccinegroupAntigen164.63±3.64184.29±1.60a196.92±1.17a176.35±1.38a165.21±3.54ConA164.60±0.99192.85±1.93a206.06±1.47a176.42±0.16a168.28±0.71rBb-TSOL18vaccinegroupSupernatant158.11±3.48178.36±1.39a193.91±1.32a173.82±1.57a154.62±1.99Antigen164.82±4.10184.82±1.53a196.89±1.45a175.87±2.05a165.17±3.50ConA165.62±3.77192.62±1.94a205.97±3.07a178.00±1.55a167.23±2.17MRScontrolgroupSupernatant152.60±1.61153.60±3.46152.16±0.87154.39±1.12156.28±1.17Antigen161.32±2.16160.32±2.98160.90±2.49160.92±1.62161.83±3.18ConA163.67±2.26163.67±2.37164.70±2.44165.74±0.80164.57±1.52

GroupStimulantionWeek02468rBb-TSO45W-4B-TSOL18Supernatant18.60±0.2721.45±0.07ab22.64±0.15ab24.72±0.21ab21.51±0.31abvaccinegroupAntigen19.44±0.0622.84±0.14ab23.65±0.37ab25.38±0.31ab22.76±0.22abConA20.58±0.1923.88±0.09ab24.54±0.29ab26.35±0.24ab23.78±0.23abrBb-TSO45W-4BSupernatant17.15±0.1219.80±0.14a21.60±0.23a23.53±0.28a21.73±0.29avaccinegroupAntigen18.70±0.1920.33±0.20a22.66±0.34a24.21±0.14a22.78±0.12aConA19.52±0.2521.73±0.29a23.36±0.28a25.81±0.20a23.44±0.35arBb-TSOL18vaccinegroupSupernatant17.47±0.2619.47±0.26a21.65±0.31a23.68±0.31a21.56±0.36aAntigen18.62±0.3020.62±0.30a22.34±0.22a24.61±0.17a22.53±0.42aConA19.59±0.3721.59±0.37a23.43±0.31a25.71±0.39a23.41±0.32aMRScontrolgroupSupernatant17.95±0.7618.20±0.8919.13±0.4120.29±0.4518.54±0.38Antigen19.43±0.9219.93±0.5820.07±0.3621.12±0.1919.53±0.20ConA21.58±0.7522.08±0.7620.97±0.4322.47±0.3221.49±0.23

GroupStimulantionWeek02468rBb-TSO45W-4B-TSOL18Supernatant39.74±0.0939.84±0.1047.55±0.19ab46.63±0.06ab39.53±0.47vaccinegroupAntigen41.28±0.0841.35±1.1148.52±0.15ab47.20±0.07ab41.26±0.09ConA42.46±0.0642.39±0.0452.64±0.21ab48.18±0.11ab42.49±0.04rBb-TSO45W-4BSupernatant39.49±0.7639.74±0.2746.52±0.21a45.21±0.20a39.43±0.29vaccinegroupAntigen41.71±0.5341.64±0.3347.51±0.25a46.52±0.17a41.19±0.40ConA42.60±0.2342.57±0.1049.56±0.19a47.57±0.38a41.96±0.54rBb-TSOL18vaccinegroupSupernatant39.37±0.1838.37±0.1846.53±0.34a45.33±0.18a39.39±0.26Antigen41.39±0.4141.39±0.4147.84±0.24a46.37±0.25a41.03±0.66ConA43.05±1.1542.63±0.2349.15±0.08a47.67±0.22a41.86±0.78MRScontrolgroupSupernatant39.33±0.2739.33±0.2738.61±0.1739.42±0.1539.32±0.15Antigen41.43±0.4041.43±0.4039.28±0.3140.42±0.2740.64±0.23ConA42.73±0.2842.73±0.2842.55±0.2541.46±0.3641.31±0.30

GroupStimulantionWeek02468rBb-TSO45W-4B-TSOL18Supernatant70.62±0.1978.29±0.06ab83.64±0.09ab85.33±0.34ab92.45±0.27abvaccinegroupAntigen72.52±0.3881.19±0.15ab85.34±0.05ab86.39±0.30ab93.55±0.28abConA73.29±0.1982.34±0.35ab86.40±0.18ab88.69±0.21ab95.64±0.14abrBb-TSO45W-4BSupernatant70.54±0.2274.40±0.27a76.64±0.17a81.46±0.34a85.66±0.20avaccinegroupAntigen72.47±0.2176.33±0.17a78.20±0.15a83.39±0.40a86.28±0.15aConA73.35±0.2078.43±0.30a79.57±0.36a84.38±0.21a88.43±0.41arBb-TSOL18vaccinegroupSupernatant70.54±0.2074.39±0.27a76.65±0.17a81.46±0.33a85.67±0.19aAntigen72.38±0.2976.38±0.29a78.42±0.27a83.47±0.35a86.64±0.28aConA73.87±0.1378.87±0.13a79.51±0.19a84.35±0.38a88.42±0.36aMRScontrolgroupSupernatant70.42±0.3672.42±0.3672.49±0.2072.43±0.2272.79±0.80Antigen72.38±0.3775.38±0.3776.34±0.3276.56±0.3975.44±0.03ConA73.51±0.3676.51±0.3678.17±0.1877.50±0.2876.94±0.60

注：與MRS對(duì)照組比較，aP<0.05；與重組Bb-TSO45W-4B疫苗組和重組Bb-TSOL18疫苗組比較，bP<0.05。

Note: Compared with MRS control group,aP<0.05; Compared with rBb-TSO45W-4B vaccine group and rBb-TSOL18 vaccine group,bP<0.05.

3討論

Bueno等(2000)發(fā)現(xiàn)囊蟲(chóng)病患者腦脊液、血清和唾液中存在特異性IgG、IgA和IgE；方文等[11]發(fā)現(xiàn)囊蟲(chóng)病豬肝、腦、骨骼肌和心肌IL-6、IL-8、TNF-α和sIL-2R升高；葉紅等[12]發(fā)現(xiàn)腦囊蟲(chóng)病患者外周血IL-4、IL-10升高，IL-2和IFN-γ降低，丙硫咪唑治療后IL-4、IL-10降低，IL-2、IFN-γ升高。胡守鋒等[13]發(fā)現(xiàn)囊蟲(chóng)病患者外周血CD4+升高，CD8+降低，CD4+/CD8+升高。上述資料表明宿主在防御囊蟲(chóng)病的過(guò)程中不僅有體液免疫反應(yīng)參與，而且有Th1、Tp和細(xì)胞免疫反應(yīng)參與，這就為我們研制豬帶絳蟲(chóng)疫苗提供了理論依據(jù)。

TSO45W-4B和TSOL18具有較強(qiáng)的免疫原性和免疫保護(hù)性[14-16]，我們研制的豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗，動(dòng)物實(shí)驗(yàn)證實(shí)，以該疫苗1011CFU口服灌胃免疫仔豬，豬帶絳蟲(chóng)卵攻擊后，可誘導(dǎo)仔豬獲得83.09%的減蚴率，具有一定的保護(hù)性[17]。本研究進(jìn)一步將豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗免疫仔豬后，分別從體液免疫、細(xì)胞免疫和細(xì)胞因子的變化觀察其免疫效果。結(jié)果表明，豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗免疫后，一方面刺激仔豬產(chǎn)生了相應(yīng)的抗體，表現(xiàn)為血清IgG、IgG1、IgG2a水平明顯升高，提示該疫苗在仔豬體內(nèi)有效地誘導(dǎo)了體液免疫反應(yīng)；另一方面刺激PBMC發(fā)生了顯著增殖，且CD4+和CD8+T細(xì)胞亞群亦呈增高趨勢(shì)，提示該疫苗在仔豬體內(nèi)有效地誘導(dǎo)了細(xì)胞免疫反應(yīng)；再者，刺激PBMC分泌了高水平的IL-2、IFN-γ、IL-4及IL-10。鑒于IgG1和IgG2a抗體亞類作為免疫反應(yīng)類型的指征，其中Th1細(xì)胞分泌的IL-2、IFN-γ有利于補(bǔ)體結(jié)合IgG2a的合成，主要介導(dǎo)細(xì)胞免疫反應(yīng)，而產(chǎn)生IL-4、IL-10的Tp細(xì)胞參與輔助B細(xì)胞刺激IgG1的生成，主要介導(dǎo)體液免疫反應(yīng)[18]。因此我們推測(cè)豬帶絳蟲(chóng)重組Bb-TSO45W-4B-TSOL18疫苗在仔豬體內(nèi)誘導(dǎo)產(chǎn)生了Th1型和Tp型的免疫反應(yīng)。

本實(shí)驗(yàn)還發(fā)現(xiàn)，TSO45W-4B-TSOL18融合抗原疫苗的免疫效果優(yōu)于TSO45W-4B或TSOL18單一抗原疫苗，可能與2種抗原融合后，綜合了這2種抗原分子的協(xié)同作用有關(guān)，提示多抗原、多表位、多基因融合的復(fù)合多價(jià)疫苗可能會(huì)提高免疫效果，或許是未來(lái)抗囊蟲(chóng)病疫苗研究的一個(gè)方向。

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DOI：10.3969/j.issn.1002-2694.2016.04.008

通訊作者：周必英，Email:zbyzl01@126.com

中圖分類號(hào)：R383.32

文獻(xiàn)標(biāo)識(shí)碼：A

文章編號(hào)：1002-2694(2016)04-0356-07

Corresponding author:Zhou Bi-ying, Email: zbyzl01@126.com

收稿日期：2015-08-03修回日期：2015-11-21

by immunization with recombinant bifidobacterium(Bb)-TSO45W-4B-TSOL18 vaccine ofTaeniasolium

WANG Ling-jun1, LIU Mei-chen1, ZHOU Ling2, YANG Feng-jiao1,JIA Qi1, JIANG Nan1, ZHOU Bi-ying1

(1.DepartmentofParasitology,ZunyiMedicalCollege,Zunyi563000,China;2.DepartmentofTraditionalChineseMedicineandAnorectum,theAffiliatedHospital,ZunyiMedicalCollege,Zunyi563000,China)

Abstract:To study dynamical changes of immune responses induced in piglets by immunization with recombinant bifidobacterium (Bb)-TSO45W-4B-TSOL18 vaccine of Taenia solium, sixteen healthy piglets of 40 days old were randomly divided into four groups: rBb-TSO45W-4B-TSOL18 vaccine group, rBb-TSO45W-4B vaccine group, rBb-TSOL18 vaccine group, and Bb liquid medium (MRS) control group. All vaccine groups were given 1011CFU through intragastric immunization. A total of two times of immunization were conducted, once for every two weeks. On 0th, 2nd, 4th, 6th and 8th week after immunization, the blood from precaval vein were collected to separate serum and peripheral blood lymphocytes (PBMC). Levels of IgG, IgG1 and IgG2a was detected by enzyme linked immunosorbent assay(ELISA). Proliferation level of PBMC was detected by methyl thiazolyl tetrazolium (MTT) method. Levels of IL-2, IFN-γ, IL-4 and IL-10 in PBMC culture supernatant were determined by ELISA. Percentage of CD4+ and CD8+ T cell subsets were detected by flow cytometry (FCM). The study demonstrated that levels of IgG, IgG1 and IgG2a in all vaccine groups increased from 2 to 8 weeks after immunization, and reached the highest level by the 4th week, 6th week, and 4th week. Proliferation level of PBMC increased from 2 to 8 weeks, and reached the highest level by the 6th week. Percentage of CD4+ and CD8+ T cells increased from 2 to 8 weeks, and reached the highest level by the 8th week and 6th week. Levels of IL-2, IFN-γ, IL-4 and IL-10 in PBMC culture supernatant increased from 2 to 6 weeks, 2 to 8 weeks, 4 to 6 weeks and 2 to 8 weeks, and reached the highest level by the 4th week, 6th week, 4th week and 8th week. The differences of above indexes between all vaccine groups and MRS control group were statistically significant. The differences between rBb-TSO45W-4B-TSOL18 vaccine group and rBb-TSO45W-4B vaccine group or rBb-TSOL18 vaccine group were statistically significant. It was concluded that rBb-TSO45W-4B-TSOL18 vaccine of Taenia solium might induce piglets to produce specific immune responses, and the immune effect of TSO45W-4B-TSOL18 fusion antigen vaccine is better than that of TSO45W-4B or TSOL18 single antigen vaccine.

Keywords:Taenia solium; recombinant Bb-TSO45W-4B-TSOL18 vaccine; immune responses; piglets

國(guó)家自然科學(xué)基金項(xiàng)目(No.81160206)資助