CAJ | 학술논문

目的构建周期型马来丝虫(Bm)半胱氨酸蛋白酶抑制剂(CPI)和3-磷酸甘油醛脱氢酶(GAPDH)重组复合基因真核表达质粒,观察其在小鼠体内的细胞免疫应答效应.方法将重组质粒pGEM-T/BmCPI和pGEM-T/BmG APDH以及真核重组表达载体分别进行双酶切,构建真核重组表达质粒pcDNA3.1 (+)-BmCPI/BmGAPDH.将纯化的pcDNA3.1(+)-BmCPI/BmGAPDH和含非甲基化胞嘧啶和鸟嘌呤的寡聚脱氧核苷酸(CpG ODN)免疫BALB/c小鼠,并设PBS对照组及空质粒对照组,共免疫3次,每次间隔2周.采用RT-PCR方法检测肌肉组织内的目的基因,采用四甲基偶氮唑盐( MTT)法和ELISA法分别检测免疫小鼠T淋巴细胞刺激增殖指数和血清中IFN-γ、IL-4水平.统计学分析采用t检验.结果 pcDNA3.1 (+)-BmCPI/BmGAPDH复合基因真核表达载体免疫小鼠后,从小鼠肌肉组织内扩增出目的基因.免疫6周后,pcDNA3.1(+)-BmCPI/CpG组和pcDNA3.1(+)-BmCPI/BmGAPDH/CpG组T淋巴细胞刺激增殖指数分别为1.466±0.635和1.610±0.112,显著高于PBS组的1.004±0.019和pcDNA3.1(+)-CpG组的1.078±0.129(t=64.438、45.318、42.749、34.314,均P＜0.05).免疫4周后,pcDNA3.1(+)-BmCPI/BmGAPDH/CpG组IFN-γ、IL-4水平均高于pcDNA3.1(+)-CpG组(t=288.053、76.453,均P＜0.05),其中IFN-γ水平与pcDNA3.1(+)-BmCPI/CpG组相比也有明显提高(t=129.642,P＜0.05).结论 pcDNA3.1(+)-BmCPI/BmGAPDH重组复合基因真核表达载体能在小鼠体内表达,并可有效诱导特异性细胞免疫应答.
목적 구건주기형마래사충(Bm)반광안산단백매억제제(CPI)화3-린산감유철탈경매(GAPDH)중조복합기인진핵표체질립,관찰기재소서체내적세포면역응답효응.방법 장중조질립pGEM-T/BmCPI화pGEM-T/BmG APDH이급진핵중조표체재체분별진행쌍매절,구건진핵중조표체질립pcDNA3.1 (+)-BmCPI/BmGAPDH.장순화적pcDNA3.1(+)-BmCPI/BmGAPDH화함비갑기화포밀정화조표령적과취탈양핵감산(CpG ODN)면역BALB/c소서,병설PBS대조조급공질립대조조,공면역3차,매차간격2주.채용RT-PCR방법검측기육조직내적목적기인,채용사갑기우담서염( MTT)법화ELISA법분별검측면역소서T림파세포자격증식지수화혈청중IFN-γ、IL-4수평.통계학분석채용t검험.결과 pcDNA3.1 (+)-BmCPI/BmGAPDH복합기인진핵표체재체면역소서후,종소서기육조직내확증출목적기인.면역6주후,pcDNA3.1(+)-BmCPI/CpG조화pcDNA3.1(+)-BmCPI/BmGAPDH/CpG조T림파세포자격증식지수분별위1.466±0.635화1.610±0.112,현저고우PBS조적1.004±0.019화pcDNA3.1(+)-CpG조적1.078±0.129(t=64.438、45.318、42.749、34.314,균P＜0.05).면역4주후,pcDNA3.1(+)-BmCPI/BmGAPDH/CpG조IFN-γ、IL-4수평균고우pcDNA3.1(+)-CpG조(t=288.053、76.453,균P＜0.05),기중IFN-γ수평여pcDNA3.1(+)-BmCPI/CpG조상비야유명현제고(t=129.642,P＜0.05).결론 pcDNA3.1(+)-BmCPI/BmGAPDH중조복합기인진핵표체재체능재소서체내표체,병가유효유도특이성세포면역응답.
Objective To construct the eukaryotic expression plasmids containing cysteine protease inhibitor (CPI) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene from periodic Brugia malayi (Bm),and to observe its cellular immune response in mouse.Methods pcDNA3.1 (+)-BmCPI/BmGAPDH was constructed.The recombinant plasmids were screened and identified by digestion with restriction enzyme.BALB/c mice were injected intramuscularly with a dosage of 100 μg purified recombinant plasmid DNA with GpG oligodeoxynucleotide (CpG ODN) and two same doses were administrated at 2-week intervals.pcDNA3.1 (+) and phosphate buffered solution (PBS) were used as controls.The tissue of muscles at 4 weeks after the third injection was collected and the target gene was detected by reverse transcription-polymerase chain reaction (RTPCR).Two weeks after the third immunization,the stimulation index (SI) of spleen lymphocytes of immunized mice was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method and the serum levels of interleukin (IL)-4 and interferon (IFN)-γ were detected by enzyme-linked immunosorbent assay (ELISA).The data were analyzed by t test.Results BmCPI/BmGAPDH gene in the injected muscle of the immunized mice was detected by RT-PCR. At 6 weeks after immunization,the SIot spleen T lymphocytes in pcDNA3.1 (+)-BmCPI/CpG group and pcDNA3.1 (+)-BmCPI/BmGAPDH/CpG group were 1.466 ± 0.635 and 1.610 ± 0.112,respectively,which were both higher than PBS group and pcDNA3.1( +)-CpG group (1.004 ± 0.019 and 1.078 ± 0.129,respectively) (t=64.438,45.318,42.749 and 34.314,respectively; all P＜0.05).At 4 weeks after immunization,the serum levels of IL-4 and IFN-γ of mice in pcDNA3.1 ( + )-BmCPI/BmGAPDH/ CpG group were significantly higher than those in pcDNA3.1 (+)-CpG group (t=288.053 and 76.453,respectively; both P＜0.05),while the serum level of IFN-γ was also higher than that in pcDNA3.1 (+)-BmCPI/CpG group (t=129.642,P＜0.05). Conclusion The recombinant eukaryotic plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH could be expressed in mice,and could elicit specific cellular immune responses in immunized mice.