【摘要】：目的:構(gòu)建日本血吸蟲(chóng)組織蛋白酶B(Sjcb2)原核表達(dá)載體pWR450-1/sjcb2并表達(dá);構(gòu)建真核表達(dá)載體pcDNA3.1(+)/sjcb2,轉(zhuǎn)染HeLa細(xì)胞,并將pcDNA3.1(+)/sjcb2直接肌注BALB/c小鼠,觀察其所誘導(dǎo)的體液免疫和細(xì)胞免疫應(yīng)答水平,初步研究該核酸疫苗對(duì)BALB/c小鼠誘導(dǎo)的免疫保護(hù)性作用,為研制新型抗日本血吸蟲(chóng)感染核酸疫苗提供實(shí)驗(yàn)依據(jù)。 方法:用PRIMER5.0引物設(shè)計(jì)軟件自行設(shè)計(jì)引物,PCR法擴(kuò)增sjcb2基因片段,將PCR產(chǎn)物純化后與pUCm-T載體連接,經(jīng)藍(lán)白斑篩選、雙酶切分析和PCR鑒定后,亞克隆入pWR450-1原核表達(dá)載體及pcDNA3.1(+)真核表達(dá)載體中,經(jīng)抗生素(氨芐青霉素)篩選、雙酶切鑒定、PCR鑒定及測(cè)序鑒定后,將構(gòu)建的pWR450-1/Sjcb2重組原核表達(dá)載體轉(zhuǎn)化大腸桿菌后經(jīng)IPTG誘導(dǎo)表達(dá),SDS-PAGE及Western blotting鑒定表達(dá)情況。構(gòu)建的pcDNA3.1(+)/sjcb2重組質(zhì)粒電穿孔轉(zhuǎn)染HeLa細(xì)胞,免疫細(xì)胞化學(xué)法觀察其在真核細(xì)胞中的表達(dá)情況;將核酸疫苗pcDNA3.1(+)/Sjcb2注射到BALB/c小鼠后腿股四頭肌肌肉組織中,每2周免疫一次,共免疫3次。末次免疫后2周,用日本血吸蟲(chóng)尾蚴攻擊感染BALR/c小鼠。PCR及免疫組化法分析Sjcb2基因在小鼠體內(nèi)的穩(wěn)定性及表達(dá)情況;MTT法檢測(cè)小鼠脾淋巴細(xì)胞特異性增殖反應(yīng);ELISA雙抗體夾心法測(cè)定攻擊感染前后小鼠脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ和IL-4的水平;瓊脂雙向擴(kuò)散試驗(yàn)測(cè)定小鼠血清中Sjcb2抗體水平;攻擊感染42天后剖殺小鼠,計(jì)算小鼠所荷成蟲(chóng)對(duì)數(shù)及肝臟所荷蟲(chóng)卵數(shù)。 結(jié)果:成功構(gòu)建pWR450-1/Sjcb2重組原核表達(dá)載體,并在E.coli DH5α中表達(dá)出一分子量約為86KDa的重組融合蛋白。 成功構(gòu)建pcDNA3.1(+)/sjcb2真核表達(dá)重組體,電穿孔轉(zhuǎn)染HeLa細(xì)胞,免疫細(xì)胞化學(xué)法表明Sjcb2能在HeLa細(xì)胞胞漿中表達(dá)。將核酸疫苗pcDNA3.1(+)/Sjcb2
[Abstract]:Objective: to construct the prokaryotic expression vector pWR450-1/sjcb2 of schistosoma japonicum cathepsin B (Sjcb2), construct eukaryotic expression vector pcDNA3.1 () / sjcb2, transfect it into HeLa cells, and inject pcDNA3.1 () / sjcb2 directly into BALB/c mice, and observe the level of humoral and cellular immune response induced by pcDNA3.1 () / sjcb2. The immune protective effect of the nucleic acid vaccine on BALB/c mice was preliminarily studied, which provided experimental basis for the development of a novel nucleic acid vaccine against Schistosoma japonicum infection. Methods: the sjcb2 gene fragment was amplified by using PRIMER5.0 primer design software. The PCR product was purified and ligated with pUCm-T vector. The product was screened by blue and white spot and identified by double enzyme digestion and PCR. Subcloned into pWR450-1 prokaryotic expression vector and pcDNA3.1 () eukaryotic expression vector, screened by antibiotics (ampicillin), identified by double enzyme digestion and identified by PCR and sequencing. The recombinant prokaryotic expression vector of pWR450-1/Sjcb2 was transformed into E. coli and identified by SDS-PAGE and Western blotting induced by IPTG. The pcDNA3.1 () / sjcb2 recombinant plasmid was electroporated into HeLa cells and its expression in eukaryotic cells was observed by immunocytochemistry. The nucleic acid vaccine pcDNA3.1 () / Sjcb2 was injected into the quadriceps muscle of the hind leg of BALB/c mice and immunized every 2 weeks. Total immunization 3 times. 2 weeks after the last immunization, The stability and expression of Sjcb2 gene in mice infected with Schistosoma japonicum cercariae in vivo were analyzed by .PCR and immunohistochemical method. The specific proliferative reaction of spleen lymphocytes in mice was detected by MTT assay and the attack was detected by Elisa double antibody sandwich method. The levels of IFN- 緯 and IL-4 in the culture supernatant of spleen lymphocytes of mice before and after infection; Agar bidirectional diffusion test was used to determine the level of Sjcb2 antibody in serum of mice, and after 42 days of infection, mice were killed to calculate the logarithm of adult worms and the number of eggs loaded by liver. Results: the recombinant prokaryotic expression vector of pWR450-1/Sjcb2 was successfully constructed, and a recombinant fusion protein with molecular weight of 86KDa was expressed in E.coli DH5 偽. The eukaryotic expression recombinant of pcDNA3.1 () / sjcb2 was successfully constructed and transfected into HeLa cells by electroporation. Immunocytochemistry showed that Sjcb2 could be expressed in the cytoplasm of HeLa cells. Nucleic acid vaccine pcDNA3.1 () / Sjcb2
【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】：2005
【分類(lèi)號(hào)】：R383

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

1 劉文琪;血吸蟲(chóng)酶類(lèi)研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));2001年05期

2 張媛;林瑞慶;李曉燕;趙光輝;;抗日本血吸蟲(chóng)藥物的研究進(jìn)展[J];中國(guó)畜牧獸醫(yī);2009年07期

3 肖西志,于三科,林矯矯,顧越星,劉金明,張亮,沈陽(yáng);應(yīng)用重組日本血吸蟲(chóng)組織蛋白酶L診斷日本血吸蟲(chóng)病的研究[J];中國(guó)預(yù)防獸醫(yī)學(xué)報(bào);2003年05期

相關(guān)碩士學(xué)位論文 前1條

1 劉瓊;豬囊尾蚴半胱氨酸蛋白酶TsCL-1基因的表達(dá)[D];新疆農(nóng)業(yè)大學(xué);2008年

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本文編號(hào)：2169635