CAJ | 학술논문

目的构建幽门螺杆菌脂蛋白Lpp20基因的真核表达载体pcDNA3.1(+)/Lpp20,并在HeLa细胞中进行表达.通过肌肉注射免疫C57BL/6小鼠,观察其诱导小鼠产生的体液免疫和细胞免疫应答水平.方法用PCR法扩增Lpp20全基因,再将Lpp20基因克隆至pcDNA3.1(+)真核细胞表达载体构建pcDNA3.1(+)/Lpp20重组体,观察其在HeLa细胞中的表达.将核酸疫苗PcDNA3.1(+)/Lpp20、对照空质粒pcDNA3.1(+)及PBS分组通过肌肉注射免疫6周龄C57BL/6小鼠.隔2周免疫一次,共免疫4次.间接ELISA法测定小鼠血清中抗Lpp20 IgG抗体水平,双抗体夹心ELISA法检测脾淋巴细胞培养上清中IFN-γ水平,MTT比色法检测脾淋巴细胞增殖反应.通过PCR法检测小鼠肌细胞中Lpp20基因的存在.结果小鼠接种pcDNA3.1(+)/Lpp20核酸疫苗后能产生特异性IgG抗体,6周后ELISA测定血清抗体A450值为0.74,效价为1:1024.核酸疫苗免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ含量明显升高[(410.36±56.23)ps/ml],与空质粒组[(25.26±10.85)pg/ml]之间差异有统计学意义(P<0.01).脾淋巴细胞增殖反应测定,核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数(2.37±0.22)明显高于空质粒组(1.53±0.47)和PBS组(1.20±0.13),P<0.01.PCR检测Lpp20基因可在小鼠肌细胞中存在.结论成功构建了pcDNA3.1(+)/Lpp20核酸疫苗,且其在小鼠体内可诱导较强的特异性体液免疫和细胞免疫应答.为进一步研究该疫苗的免疫保护作用提供实验依据.
목적 구건유문라간균지단백Lpp20기인적진핵표체재체pcDNA3.1(+)/Lpp20,병재HeLa세포중진행표체.통과기육주사면역C57BL/6소서,관찰기유도소서산생적체액면역화세포면역응답수평.방법 용PCR법확증Lpp20전기인,재장Lpp20기인극륭지pcDNA3.1(+)진핵세포표체재체구건pcDNA3.1(+)/Lpp20중조체,관찰기재HeLa세포중적표체.장핵산역묘PcDNA3.1(+)/Lpp20、대조공질립pcDNA3.1(+)급PBS분조통과기육주사면역6주령C57BL/6소서.격2주면역일차,공면역4차.간접ELISA법측정소서혈청중항Lpp20 IgG항체수평,쌍항체협심ELISA법검측비림파세포배양상청중IFN-γ수평,MTT비색법검측비림파세포증식반응.통과PCR법검측소서기세포중Lpp20기인적존재.결과 소서접충pcDNA3.1(+)/Lpp20핵산역묘후능산생특이성IgG항체,6주후ELISA측정혈청항체A450치위0.74,효개위1:1024.핵산역묘면역조소서비림파세포경특이성항원자격후,배양상청중IFN-γ함량명현승고[(410.36±56.23)ps/ml],여공질립조[(25.26±10.85)pg/ml]지간차이유통계학의의(P<0.01).비림파세포증식반응측정,핵산역묘조소서비림파세포경특이성항원자격후,자격지수(2.37±0.22)명현고우공질립조(1.53±0.47)화PBS조(1.20±0.13),P<0.01.PCR검측Lpp20기인가재소서기세포중존재.결론 성공구건료pcDNA3.1(+)/Lpp20핵산역묘,차기재소서체내가유도교강적특이성체액면역화세포면역응답.위진일보연구해역묘적면역보호작용제공실험의거.
Objective To construct an eukaryotic expression plasmid PeDNA3.1 (+)/Lpp20 and to detect its expression in HeLa cells, and to observe the humoral and cellular immune responses in C57BL/6 mice induced by the Helicobacter pylori Lpp20 DNA vaccine injected intramuscularly. Methods The Lpp20 gene was amplified by PCR. PCR product was subcloned into the eukaryotic expression vector pcDNA3.1 (+)/ Lpp20, and the recombinant plasmid was transfected into HeLa cells using Liposome. After verifying that the Lpp20 antigen gene could be expressed in HeLa cells. Six weeks old C57BL/6 mice were immunized with pcDNA3.1 (+)/Lpp20 or pcDNA3.1 (+) or PBS buffer intramuscularly at 2-week interval for four times. ELISA was used for the quantitative detection of the specific IgG antibody in the sera of C57BL/6 mice and the cytokine IFN-γ in mice spleen lymphocyte culture medium after stimulating by Lpp20. The proliferation response of spleen cells was detected by MTT assay. The Lpp20 gene in muscle was identified by PCR. Results The significant specific antibody titers were detected by ELISA in DNA vaccine groups and the highest titer was 1:1024 after 6 weeks. The cytnkine IFN-γ in mice inoculated with pcDNA3.1 (+)/Lpp20 was increased and reached (410.36±56.23) pg/ml. A significant difference was tested between the experiment group and the control group[(25.26±10.85)pg/ml] ,P <0.01. The proliferation response of spleen cells of DNA vaccine group(SI: 2.37±0.22) was significantly higher than those of mice injected with pcDNA3.1 (+) (SI:1.53+0.47) ,P<0.01. Lpp20 gene could exist constantly in musculature cells of mice. Conclusion The eukaryotic expression recombinant pcDNA3.1 (+)/Lpp20 was successfully constructed. Strong humoral and cellular im-munity can be induced by DNA vaccine of pcDNA3.1(+)/Lpp20 in C57BL/6 mice, which might be helpful for further investigation concerning the immunoprotection of DNA vaccine.