豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗誘導仔豬免疫應答的動態觀察

王靈軍，劉美辰，周　泠，楊鳳嬌，賈　啟，江　楠，周必英

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豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗誘導仔豬免疫應答的動態觀察

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.遵義醫學院寄生蟲學教研室，遵義563000；2.遵義醫學院附屬醫院中醫肛腸科，遵義563000

摘要：目的研究豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗免疫仔豬后誘導免疫應答的動態變化。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基(MRS)對照組。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胞(PBMC)。采用ELISA法檢測仔豬血清IgG、IgG1及IgG2a水平和PBMC培養上清液IL-2、IFN-γ、IL-4及IL-10水平；MTT比色法檢測PBMC增殖水平；FCM檢測CD4+和CD8+T細胞亞群。結果各疫苗組血清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周達較高水平；CD4+和CD8+T細胞水平均在免疫后2～8周升高，均在免疫后8周和6周達較高水平；PBMC培養上清液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周達較高水平。各疫苗組上述指標與MRS對照組比較差異均有統計學意義(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計學意義(P<0.05)。結論豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗免疫仔豬可誘導產生特異性的免疫應答，且TSO45W-4B-TSOL18融合抗原疫苗組優于TSO45W-4B或TSOL18單一抗原疫苗組。

關鍵詞：豬帶絳蟲；重組Bb-TSO45W-4B-TSOL18疫苗；免疫應答；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)俗稱囊蟲病，是由豬帶絳蟲(Taeniasolium)幼蟲-囊尾蚴引起的一種危害嚴重的人獸共患寄生蟲病。藥物及手術治療都有其局限性，有保護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，已在細菌、病毒、腫瘤、寄生蟲等領域得到了廣泛應用[3]。本研究擬在成功構建豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗[4-5]的基礎上，以該疫苗免疫仔豬，動態觀察其誘導的免疫應答。

1材料與方法

1.1疫苗來源豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗、重組Bb-TSO45W-4B疫苗、重組Bb-TSOL18疫苗由本室構建保存[4,6-7]。

1.2實驗動物40 d齡健康仔豬16頭，每頭體重約15 kg，購自遵義縣三盆鎮養殖場，由遵義醫學院實驗動物中心飼養管理。1.3主要試劑與儀器純化的豬帶絳蟲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Southern Biotech公司)；IL-2、IFN-γ、IL-4和IL-10試劑盒[美國rndsystem(RD)北京永輝生物技術有限公司]；異硫氰酸熒光素(FITC)標記CD4+單抗和藻紅蛋白(PE)標記CD8+單抗(美國BD Biosciences公司)；ConA(美國Sigma公司)；RPMI1640培養基、FBS、MTT、DMSO(上海生工生物技術有限公司)；豬PBMC分離液(大連寶生物公司)。酶標儀(山東高密彩虹分析儀器有限公司)；流式細胞儀由遵義醫學院免疫學教研室提供；37 ℃恒溫水浴箱(上海市躍進農場醫療器械廠)。

1.4動物分組與免疫將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于無菌EDTA抗凝管，用10% FBS的RPMI1640培養液稀釋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培養液稀釋5倍，洗滌2次，即為淋巴細胞，細胞沉淀用RPMI1640培養液懸浮后，用臺盼藍檢測細胞活力和計數，計數后，用RPMI1640培養液調至為5×106/mL。

1.7PBMC增殖反應取出細胞培養板，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44 h，加入10 μL MTT(5 mg/mL)，繼續培養4 h，控干，加入100 μL DMSO，吹打溶解，立即用酶標儀測定OD630nm值。

1.8T細胞亞群檢測吸取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1次，棄上清，PBS懸浮，用流式細胞儀檢測PBMC中CD4+和CD8+T細胞亞群百分率。

2結果

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周達較高水平，與MRS對照組比較，差異均具有統計學意義(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，差異無統計學意義(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，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周達較高水平，與MRS對照組比較，差異均具有統計學意義(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，差異無統計學意義(P>0.05)。用Ag或ConA刺激時，PBMC增殖水平類似于不加刺激物結果，見表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細胞水平均在免疫后2～8周升高，均在免疫后8周、6周達較高水平，與MRS對照組比較，差異均具有統計學意義(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，差異無統計學意義(P>0.05)，見表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，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周達到較高水平，與MRS對照組比較，差異均具有統計學意義(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，差異無統計學意義(P>0.05)。用Ag或ConA刺激時，PBMC培養上清液IL-2、IFN-γ、IL-4及IL-10水平類似于不加刺激物結果，見表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)發現囊蟲病患者腦脊液、血清和唾液中存在特異性IgG、IgA和IgE；方文等[11]發現囊蟲病豬肝、腦、骨骼肌和心肌IL-6、IL-8、TNF-α和sIL-2R升高；葉紅等[12]發現腦囊蟲病患者外周血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]發現囊蟲病患者外周血CD4+升高，CD8+降低，CD4+/CD8+升高。上述資料表明宿主在防御囊蟲病的過程中不僅有體液免疫反應參與，而且有Th1、Tp和細胞免疫反應參與，這就為我們研制豬帶絳蟲疫苗提供了理論依據。

TSO45W-4B和TSOL18具有較強的免疫原性和免疫保護性[14-16]，我們研制的豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗，動物實驗證實，以該疫苗1011CFU口服灌胃免疫仔豬，豬帶絳蟲卵攻擊后，可誘導仔豬獲得83.09%的減蚴率，具有一定的保護性[17]。本研究進一步將豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗免疫仔豬后，分別從體液免疫、細胞免疫和細胞因子的變化觀察其免疫效果。結果表明，豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗免疫后，一方面刺激仔豬產生了相應的抗體，表現為血清IgG、IgG1、IgG2a水平明顯升高，提示該疫苗在仔豬體內有效地誘導了體液免疫反應；另一方面刺激PBMC發生了顯著增殖，且CD4+和CD8+T細胞亞群亦呈增高趨勢，提示該疫苗在仔豬體內有效地誘導了細胞免疫反應；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類型的指征，其中Th1細胞分泌的IL-2、IFN-γ有利于補體結合IgG2a的合成，主要介導細胞免疫反應，而產生IL-4、IL-10的Tp細胞參與輔助B細胞刺激IgG1的生成，主要介導體液免疫反應[18]。因此我們推測豬帶絳蟲重組Bb-TSO45W-4B-TSOL18疫苗在仔豬體內誘導產生了Th1型和Tp型的免疫反應。

本實驗還發現，TSO45W-4B-TSOL18融合抗原疫苗的免疫效果優于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種抗原分子的協同作用有關，提示多抗原、多表位、多基因融合的復合多價疫苗可能會提高免疫效果，或許是未來抗囊蟲病疫苗研究的一個方向。

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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：R383.32

文獻標識碼：A

文章編號：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

國家自然科學基金項目(No.81160206)資助