CpG ODN、氢氧化铝和大肠埃希菌不耐热肠毒素无毒突变体联合佐剂对猪轮状病毒△VP8亚蛋白免疫效果的影响  

Influence of CpG ODN, aluminum hydroxide and avirulent E. coli heatlabile enterotoxin mutant combined adjuvant on immune effect of porcine rotavirus △VP8* subunit vaccine

作　　者：冉旭华[1] 魏晓曼[2] 闻晓波[1] 曹思[1] 张峣[1] 崔玉东[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院预防兽医学省重点实验室,黑龙江大庆163319 [2]香港浸会大学常熟研究院,江苏苏州215000

出　　处：《中国生物制品学杂志》2015年第8期780-785,790,共7页Chinese Journal of Biologicals

基　　金：国家自然科学青年基金项目(31201909);黑龙江博士后科研启动资助项目(LBH-Q13134);黑龙江省自然科学基金(QC2013C028;C2015042)

摘　　要：目的评价Cp G ODN、氢氧化铝[Al(OH)3]和大肠埃希菌不耐热肠毒素(heat-labile enterotoxin,LT)无毒突变体联合佐剂对猪轮状病毒(porcine rotavirus,PRV)△VP8*亚蛋白免疫效果的影响。方法将LT A亚基上63位丝氨酸突变为赖氨酸,构建LT的无毒突变体重组表达质粒p ET-30a(+)-LTK63,于E.coli表达重组蛋白LTK63,并进行纯化。将小鼠随机分为LTK63+Al(OH)3组、Cp G ODN+Al(OH)3组、Cp G ODN+LTK63+Al(OH)3组及Al(OH)3对照组,将P2-SB-1AΔVP8*抗原蛋白分别与各组相应佐剂共同免疫小鼠,均经颈部皮下注射,隔2周免疫1次,共3次,于每次免疫后14 d采血,分离血清,间接ELISA法检测血清抗体效价及Ig G亚型;末次免疫2周后,MTT法检测小鼠特异性淋巴细胞的增殖。结果经双酶切及测序鉴定,重组质粒p ET-30a(+)-LTK63构建正确;表达和纯化产物经SDS-PAGE及Western blot分析,均可见相对分子质量约33 000(A亚基)和13 000(B亚基)的目的蛋白条带。二免后,Cp G ODN+LTK63+Al(OH)3组的抗体滴度显著高于LTK63+Al(OH)3组、Cp G ODN+Al(OH)3组和Al(OH)3对照组(P<0.05);末次免疫后2周,Cp G ODN+Al(OH)3组抗体滴度显著高于Al(OH)3对照组、Cp G ODN+LTK63+Al(OH)3组和LTK63+Al(OH)3组(P<0.001);Cp G ODN+LTK63+Al(OH)3和LTK63+Al(OH)3组小鼠血清中Ig G2a和Ig G1水平相当,Cp G ODN+Al(OH)3组血清抗体以Ig G2a为主,Al(OH)3对照组血清Ig G以Ig G1为主;Cp G ODN+Al(OH)3组小鼠特异性淋巴细胞的增殖能力最强。结论 Cp G ODN+Al(OH)3联合佐剂表现出明显的协同效应,可显著提高小鼠对PRV△VP8*亚蛋白的体液免疫和细胞免疫应答。Objective To evaluate the influence of Cp G ODN, aluminum hydroxide and E. coli heat-labile enterotoxin（LT）mutant on the immune effect of porcine rotavirus △VP8＊ subunit vaccine. Methods The serine at site 63 of LT subunit A was mutanted into lysine. The recombinant plasmid p ET-30a（＋）-LTK63 containing the obtained avirulent mutant gene was constructed and transformed to E. coli DH5α, and the expressed LTK63 protein was purified. BALB / c mice were randomly divided into four groups, and immunized s.c. with recombinant P2-SB-1A△VP8＊protein mixed with LTK63 ＋ Al（OH）3, Cp G ODN ＋ Al（OH）3, Cp G ODN ＋ LTK63 ＋ Al（OH）3and Al（OH）3respectively, once 2 weeks for 3times. Serum samples were collected 14 d after each immunization and determined for antibody titer and Ig G subtype by indirect E LISA. The proliferation of specific lymphocytes was determined by MTT assay 2 weeks after the last immunization. Results Restriction analysis and sequencing proved that recombinant plasmid p ET-30a（＋）-LTK63 was constructed correctly. Subunits A and B, with relative molecular masses of about 33 000 and about 13 000 respectively,were shown on SDS-PAGE and Western blot profiles of expressed and purified products. The antibody titer in Cp G ODN ＋LTK634 ＋ Al（OH）3group after the second immunization was significantly higher than those in LTK63 ＋ Al（OH）3, Cp G ODN ＋ Al（OH）3and Al（OH）3groups（P 〈 0. 05）. However, the antibody titer 2 weeks after the last immunization in Cp G ODN ＋ Al（OH）3group was significantly higher than those in Al（OH）3, Cp G ODN ＋ LTK63 ＋ Al（OH）3and LTK63 ＋Al（OH）3groups（P 〈 0. 001）. Both Ig G2 a and Ig G1 levels in sera of mice in Cp G ODN ＋ LTK63 ＋ Al（OH）3group were equal to those in LTK63 ＋ Al（OH）3group. The serum antibodies in Cp G ODN ＋ Al（OH）3group were mainly Ig G2 a, while those in Al（OH）3group were mainly Ig G1. The proliferation ability of specific lymphocytes in Cp G ODN ＋ Al（OH）

关 键 词：Cp G ODN 氢氧化铝 不耐热肠毒素 联合佐剂 猪轮状病毒 

分 类 号：S855.3[农业科学—临床兽医学] R392-33[农业科学—兽医学]