摘要
目的观察重组抗原Ag85b和ESAT6-CFP10(EC)联合氢氧化铝佐剂(Al)和PolyIC(IC)佐剂诱导小鼠的免疫效果及对豚鼠结核的预防作用。方法将BALB/c小鼠随机分为6组:PBS组、Pro组(Ag85b+EC)、Al+IC组、Pro+Al组(Ag85b+EC+Al)、Pro+IC组(Ag85b+EC+IC)、Pro+Al+IC组(Ag85b+EC+Al+IC),经小鼠后肢内侧肌肉注射,共注射3次,间隔10 d。末次注射后2周,摘眼球采血,分离血清,采用间接ELISA法检测IgG抗体效价;同时处死小鼠,无菌取脾,分离淋巴细胞,采用ELISPOT法检测抗原特异性的IFNγ水平。将豚鼠随机分为3组:NS组、BCG组和Pro+Al+IC组,NS组和Pro+Al+IC组免疫3次,间隔10 d;BCG组仅免疫1次,于前两组第3次免疫时进行免疫。末次免疫30 d后经皮下注射结核分枝杆菌(Mycobacterium tuberculosis,Mtb)菌液,攻毒41 d后,处死豚鼠,解剖,检测肝、脾、肺的病变程度,并进行评分,同时分离脾脏Mtb,计算每只豚鼠的脾菌分离对数值。结果经Ag85b多肽和EC多肽刺激后,Pro+Al+IC组IFNγ斑点形成细胞(spot forming cell,SFC)数均显著高于其他各组(P<0.000 1或<0.05),同组间经Ag85b多肽刺激后的SFC均高于经EC多肽刺激后的SFC。Pro+Al+IC组血清Ag85b特异性IgG抗体效价最高,与Pro+Al组比较,差异有统计学意义(P<0.05),但与Pro+IC组比较,差异无统计学意义(P>0.05);Pro+Al+IC组血清EC特异性IgG抗体效价最高,与Pro+Al和Pro+IC组比较,差异均有统计学意义(P<0.05);同组间Ag85b特异性IgG抗体效价高于EC特异性IgG抗体效价。Pro+Al+IC组豚鼠脏器综合病变指数和脾菌分离对数值均显著高于BCG组(P<0.000 1),略低于NS组,但差异无统计学意义(P>0.05)。结论氢氧化铝佐剂和PolyIC佐剂联合使用可协同增强结核分枝杆菌抗原Ag85b和EC的细胞免疫应答及体液免疫应答;但Ag85b和EC配伍构成的重组亚单位疫苗对豚鼠预防性保护力较弱,不适合用作暴露前预防。
Objective To observe the immune effect of recombinant antigens Ag85b and ESAT6-CFP10 （Pro） combined with aluminum hydroxide and PolyIC （IC） in mice and the preventive effect on tuberculosis in guinea pigs. Methods BALB/c mice were randomly divided into PBS, Pro （Ag85b ＋ EC）, A1 ＋ IC, Pro ＋ A1 （Ag85b ＋ EC ＋ A1）, Pro ＋ IC （Ag85b ＋ EC ＋ IC） and Pro ＋ A1 ＋ IC（Ag85b ＋ EC ＋ A1 ＋ IC） groups, six for each. The mice in various groups were injected i. m. for 3 times each at an interval of 10 d. Serum samples were collected 2 weeks after the last injection and determined for IgG titer by indirect ELISA. The mice were killed, of which the spleen lymphocytes were isolated aseptically and determined for IFN~/level by ELISPOT. Guinea pigs were randomly divided into normal saline （NS）, BCG and Pro ＋ A1 ＋ IC groups. The guinea pigs in NS and Pro ＋ A1 ＋ IC were immunized for 3 times each at an interval of 10 d, while those in BCG group for＇a single time at the same time of the last immunization in the other two groups. The guinea pigs in various groups were challenged s. c. with Mycobacterium tuberculosis （Mtb） 30 d after the last immunization and killed 41 d after challenge, of whichthe liver, spleen and lung were scored for lesion. Meanwhile, Mtb was isolated from spleen of each guinea pig, of which the log was calculated. Results The number of IFN^-specific spot forming cells （SFCs） in Pro ＋ A1 ＋ IC group after stimulation with Ag85b and EC were significantly larger than those in other groups （P 〈 0. 000 1 or P 〈 0. 05）. However, the numbers of SFCs after stimulation with Ag85b were larger than those with EC in the same groups. The serum specific IgG titer against Ag85b in Pro ＋ A1 ＋ IC group was the highest in various groups, which was significantly higher than that in Pro ＋ A1 group（P 〈 0. 05） while insignificantly higher than that in Pro ＋ IC group （P 〉 0. 05）. The serum specific IgG titer against EC in Pro ＋ A1 ＋ IC g
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第3期299-304,共6页
Chinese Journal of Biologicals
基金
"十二五"国家科技重大专项(2012ZX10004701)
关键词
结核疫苗
氢氧化铝
PolyIC
免疫原性
保护效力
Mycobacterium tuberculosis vaccine
Aluminum hydroxide
PolyIC
Immunogenicity
Protective efficacy
