淋球菌NspA核酸疫苗的構(gòu)建及其免疫活性的初步研究
發(fā)布時(shí)間：2019-04-12 06:39
【摘要】：目的：根據(jù)GenBank登錄的淋球菌WHO—A株的NspA核酸序列設(shè)計(jì)引物，擴(kuò)增出NspA基因，構(gòu)建真核表達(dá)載體pcDNA3.1(+)／NspA，直接肌注免疫BALB／c小鼠，觀察其所誘導(dǎo)產(chǎn)生的體液免疫和細(xì)胞免疫應(yīng)答水平，為研制新型、高效的淋球菌核酸疫苗提供實(shí)驗(yàn)依據(jù)。 方法：用PRIMER5.0軟件設(shè)計(jì)引物，PCR擴(kuò)增NspA全基因；將PCR產(chǎn)物純化后與pUCm-T載體相連，經(jīng)藍(lán)白斑篩選、雙酶切鑒定及測(cè)序鑒定后，亞克隆至pcDNA3.1(+)真核表達(dá)載體中；陽(yáng)性克隆經(jīng)雙酶切及測(cè)序鑒定后轉(zhuǎn)染RAW264.7和COS-7細(xì)胞，用RT-PCR檢測(cè)NspA mRNA的水平，免疫細(xì)胞化學(xué)法檢測(cè)蛋白表達(dá)。以pcDNA3.1(+)／NspA肌注免疫6w齡BALB／c小鼠，試管凝集法檢測(cè)免疫小鼠血清中抗NspA抗體水平，ELISA雙抗體夾心法檢測(cè)脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ水平，MTT比色法檢測(cè)脾淋巴細(xì)胞增殖反應(yīng)，PCR檢測(cè)淋球菌NspA基因在小鼠股四頭肌的存在。 結(jié)果：成功構(gòu)建了pcDNA3.1(+)／NspA真核表達(dá)載體，以脂質(zhì)體轉(zhuǎn)染RAW264.7和COS-7細(xì)胞后，用RT-PCR檢測(cè)NspA mRNA的水平，免疫細(xì)胞化學(xué)法檢測(cè)蛋白表達(dá)，表明重組質(zhì)粒能在真核細(xì)胞有效轉(zhuǎn)錄和表達(dá)NspA。小鼠接種pcDNA3.1(+)／NspA核酸疫苗后，能產(chǎn)生抗NspA的特異性抗體，其滴度隨著時(shí)間的增加和加強(qiáng)免疫而增高，6w后抗體最高滴度達(dá)1∶640。核酸疫苗免疫組小鼠脾淋巴細(xì)胞經(jīng)PHA刺激后，，培養(yǎng)上清中IFN-γ含量明顯升高(169.71±30.52pg／mL)，與空質(zhì)粒組(23.79±11.85pg／mL)和PBS組(8.71±2.50pg／mL)之間有顯著性差異(P＜0.01)。脾淋巴細(xì)胞增殖反應(yīng)測(cè)定，未加PHA時(shí)，T淋巴細(xì)胞呈單個(gè)分布，無(wú)細(xì)胞增殖；加入PHA后，核酸疫苗組細(xì)胞增殖明顯活躍，細(xì)胞成團(tuán)增長(zhǎng)，而對(duì)照組細(xì)胞增殖不
[Abstract]:Objective: according to the NspA sequence of Neisseria gonorrhoeae WHO-A strain logged in by GenBank, primers were designed to amplify NspA gene and construct eukaryotic expression vector pcDNA3.1 () / NspA, to immunize BALB/c mice by direct intramuscular injection. The humoral and cellular immune responses were observed in order to provide experimental basis for the development of a new and efficient nucleic acid vaccine of Neisseria gonorrhoeae. Methods: the whole NspA gene was amplified by PCR with PRIMER5.0 software, and the PCR product was purified and linked to pUCm-T vector. After blue and white spot screening, double enzyme digestion and sequencing identification, the product was subcloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were transfected into RAW264.7 and COS-7 cells by restriction endonuclease digestion and sequencing. The expression of NspA mRNA and protein were detected by RT-PCR and immunocytochemistry respectively. Six-week-old BALB/c mice were immunized with pcDNA3.1 () / NspA intramuscular injection. The levels of anti-NspA antibody in serum and IFN- 緯 in supernatant of spleen lymphocyte culture were detected by test tube agglutination method and ELISA double antibody sandwich method, respectively. MTT colorimetry was used to detect the proliferation of splenic lymphocytes and PCR was used to detect the presence of Neisseria gonorrhoeae NspA gene in quadriceps femoris of mice. Results: the eukaryotic expression vector of pcDNA3.1 () / NspA was constructed successfully. After transfection with liposome into RAW264.7 and COS-7 cells, the level of NspA mRNA was detected by RT-PCR and the expression of protein was detected by immunocytochemistry. The results show that the recombinant plasmid can effectively transcribe and express NspA. in eukaryotic cells. Mice inoculated with pcDNA3.1 () / NspA nucleic acid vaccine produced specific antibodies against NspA, and the titers of the antibodies increased with the increase of time and enhanced immunization. The highest titers of anti-NspA antibodies reached 1? 640 at 6 weeks after inoculation. After stimulated by PHA, the content of IFN- 緯 in the supernatant of mice immunized with nucleic acid vaccine increased significantly (169.71 鹵30.52 PG / mL),). There was a significant difference between the control group (23.79 鹵11.85 PG / mL) and the PBS group (8.71 鹵2.50pg/mL) (P < 0.01). The proliferation reaction of spleen lymphocytes showed that without PHA, T lymphocytes showed a single distribution and no cell proliferation. After adding PHA, the proliferation of T lymphocytes in nucleic acid vaccine group was obviously active and the cells in the control group did not proliferate.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
本文編號(hào)：2456786
[Abstract]:Objective: according to the NspA sequence of Neisseria gonorrhoeae WHO-A strain logged in by GenBank, primers were designed to amplify NspA gene and construct eukaryotic expression vector pcDNA3.1 () / NspA, to immunize BALB/c mice by direct intramuscular injection. The humoral and cellular immune responses were observed in order to provide experimental basis for the development of a new and efficient nucleic acid vaccine of Neisseria gonorrhoeae. Methods: the whole NspA gene was amplified by PCR with PRIMER5.0 software, and the PCR product was purified and linked to pUCm-T vector. After blue and white spot screening, double enzyme digestion and sequencing identification, the product was subcloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were transfected into RAW264.7 and COS-7 cells by restriction endonuclease digestion and sequencing. The expression of NspA mRNA and protein were detected by RT-PCR and immunocytochemistry respectively. Six-week-old BALB/c mice were immunized with pcDNA3.1 () / NspA intramuscular injection. The levels of anti-NspA antibody in serum and IFN- 緯 in supernatant of spleen lymphocyte culture were detected by test tube agglutination method and ELISA double antibody sandwich method, respectively. MTT colorimetry was used to detect the proliferation of splenic lymphocytes and PCR was used to detect the presence of Neisseria gonorrhoeae NspA gene in quadriceps femoris of mice. Results: the eukaryotic expression vector of pcDNA3.1 () / NspA was constructed successfully. After transfection with liposome into RAW264.7 and COS-7 cells, the level of NspA mRNA was detected by RT-PCR and the expression of protein was detected by immunocytochemistry. The results show that the recombinant plasmid can effectively transcribe and express NspA. in eukaryotic cells. Mice inoculated with pcDNA3.1 () / NspA nucleic acid vaccine produced specific antibodies against NspA, and the titers of the antibodies increased with the increase of time and enhanced immunization. The highest titers of anti-NspA antibodies reached 1? 640 at 6 weeks after inoculation. After stimulated by PHA, the content of IFN- 緯 in the supernatant of mice immunized with nucleic acid vaccine increased significantly (169.71 鹵30.52 PG / mL),). There was a significant difference between the control group (23.79 鹵11.85 PG / mL) and the PBS group (8.71 鹵2.50pg/mL) (P < 0.01). The proliferation reaction of spleen lymphocytes showed that without PHA, T lymphocytes showed a single distribution and no cell proliferation. After adding PHA, the proliferation of T lymphocytes in nucleic acid vaccine group was obviously active and the cells in the control group did not proliferate.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
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