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目的研究霍乱病毒B亚单位(cholera toxin subunit B,CTB)作为基因佐剂对单纯疱疹病毒2型(herpes simplex virus type 2,HSV-2)DNA疫苗pg D免疫效果的影响。方法将CTB片段克隆至真核表达质粒pc DNA3Kan(p Kan)中,构建基因佐剂pc DNA3Kan-CTB(p CTB)。将BALB/c小鼠随机分为p CTB无疫苗组、pg D/p CTB佐剂组、pg D/p Kan空载体组及PBS对照组,经后腿肌肉多点注射,共免疫3次,每次间隔2周。于末次免疫2周后,经小鼠眼球采血,分离血清,ELISA法检测HSV-2特异性Ig G水平及Ig G1、Ig G2a亚型抗体滴度;制备小鼠脾细胞,MTS法检测小鼠脾脏T细胞增殖能力;将病毒稀释后经腹腔接种小鼠,于免疫2周后,进行HSV-2致死剂量攻毒试验。结果p CTB经双酶切及测序鉴定证明构建正确;pg D/p CTB佐剂组小鼠血清中HSV-2特异性Ig G水平明显高于其他3组(P<0.001),Ig G1和Ig G2a滴度均明显高于pg D/p Kan空载体组(P<0.001),其中Ig G2a增幅更明显,Ig G2a/Ig G1比值与pg D/p Kan空载体组相比,由(1.176±0.11)增加至(1.316±0.07)(P<0.05);pg D/p CTB佐剂组小鼠脾细胞在HSV-2刺激下诱导的特异性T细胞增殖反应明显强于PBS对照组及pg D/p Kan空载体组(P<0.05);攻毒14 d内,pg D/p CTB佐剂组小鼠存活率高达100%。结论 p CTB作为基因佐剂能够显著提高pg D诱导小鼠产生抗原特异性Ig G抗体水平,增强HSV-2特异性T细胞增殖反应,并介导以细胞免疫为主的Th1/Th2混合型免疫反应。
Objective To investigate the effect of cholera toxin subunit B (CTB) as a gene adjuvant on the immunogenicity of herpes simplex virus type 2 (HSV-2) DNA vaccine pg D. Methods CTB fragment was cloned into eukaryotic expression plasmid pcDNA3Kan (p Kan) to construct gene adjuvant pcDNA3Kan-CTB (p CTB). BALB / c mice were randomly divided into p CTB vaccine-free group, pg D / p CTB adjuvant group, pg D / p Kan empty vector group and PBS control group. Interval of 2 weeks. Two weeks after the last immunization, blood was collected from the eyeballs of mice and the serum was separated. The specific Ig G of HSV-2 and the antibody titer of Ig G1 and Ig G2a subtypes were detected by ELISA. Mice splenocytes Spleen T cell proliferation ability; The virus was diluted intraperitoneally inoculated mice, 2 weeks after immunization, HSV-2 lethal dose challenge test. Results The results of double restriction enzyme digestion and sequencing confirmed that pCTB was constructed correctly. The serum level of HSV-2-specific Ig G in pg D / p CTB adjuvant group was significantly higher than that in the other three groups (P <0.001) G2a titers were significantly higher than that of pg D / p Kan empty vector group (P <0.001), and the increase of Ig G2a was more obvious. The ratio of Ig G2a / Ig G1 was (1.176 ± 0.11) to (1.316 ± 0.07) (P <0.05). The specific T cell proliferation induced by HSV-2 in pg D / p CTB adjuvant group was significantly stronger than that in PBS control group and pg D / p Kan empty vector group (P <0.05). Within 14 days after challenge, the survival rate of mice in pg D / p CTB adjuvant group was as high as 100%. Conclusion p CTB as a gene adjuvant can significantly increase the level of antigen-specific Ig G antibody induced by pg D, enhance the HSV-2-specific T cell proliferation and mediate the combination of Th1 / Th2 immune response reaction.