本文選題：剛地弓形蟲 + 熱休克蛋白70��； 參考：《山西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的 構(gòu)建弓形蟲RH株p3×Flag-CMW-14-TgHSP70真核表達(dá)重組質(zhì)粒。觀察不同劑量的重組質(zhì)粒p3×Flag-CMW-14-TgHSP70皮下肌肉注射免疫BALB/c小鼠誘導(dǎo)的細(xì)胞免疫應(yīng)答動態(tài)變化。 方法 第一部分:設(shè)計合成TgHSP70引物,PCR擴(kuò)增其基因片段,獲得弓形蟲速殖子熱休克蛋白(TgHSP70)基因編碼片段,雙酶切后與真核表達(dá)質(zhì)粒p3×Flag-CMW-14連接,連接產(chǎn)物經(jīng)酶切、PCR及測序鑒定。脂質(zhì)體法將重組體轉(zhuǎn)染HEK 293T細(xì)胞中,TgHSP70的表達(dá)產(chǎn)物通過RT-PCR和Western blot在基因和蛋白兩個水平進(jìn)行鑒定。 第二部分:125只BALB/c小鼠隨機(jī)均分5組,分別用50μg、100μg、150μg重組質(zhì)粒p3×Flag-CMW-14-TgHSP70/只皮下肌肉注射免疫小鼠3次,各間隔2周,空白對照組注射100μl Elution緩沖液,空質(zhì)粒組注射50μg p3×Flag-CMW-14,重組質(zhì)粒和空質(zhì)粒分別溶于100μl Elution緩沖液中。分別于首免后第2、4、6、8、10周摘眼球采血,分離血清,每組5只,ELISA法測定血清中IL-2、IFN-γ水平。頸椎脫臼處死小鼠,無菌取脾,分離脾淋巴細(xì)胞并計數(shù);再加入終濃度為5μg/ml的ConA,5%CO2、37℃培養(yǎng)24h、72h,取培養(yǎng)上清。ELISA法測定培養(yǎng)24h上清液中IL-4、IL-2水平和培養(yǎng)72h上清液中IFN-γ水平。 結(jié)果 PCR擴(kuò)增獲得約495 bp的HSP70編碼基因片段,p3×Flag-CMW-14-TgHSP70重組體構(gòu)建成功,陽性克隆經(jīng)雙酶切、PCR及測序鑒定,基因片段序列完全正確。將p3×Flag-CMW-14-TgHSP70真核表達(dá)重組質(zhì)粒轉(zhuǎn)染到HEK 293T細(xì)胞中,經(jīng)RT-PCR檢測可見預(yù)期大小的目的基因條帶,Western-blot檢測表達(dá)產(chǎn)物大小約19 kDa。 50μg、100μg、150μg重組質(zhì)粒組小鼠血清IL-2水平在免疫后第6周明顯上升,第8周達(dá)到頂峰,第10周稍有回落。50μg、100μg、150μg重組質(zhì)粒組和對照組的血清IFN-γ水平隨時間改變基本一致,無明顯差異。免疫后第10周,50μg重組質(zhì)粒組脾細(xì)胞數(shù)(17.362×10~6個/ml)顯著高于150μg組(15.22×10~6個/ml)、100μg組(15.66×10~6個/ml)、空質(zhì)粒組(14.82×10~6個/ml)和空白對照組(16.076×10~6個/ml)(F=4.478,P0.05)。免疫后第2～10周150μg組脾淋巴細(xì)胞IL-4水平均高于50μg和100μg組,但差異無統(tǒng)計學(xué)意義。免疫后第2～10周,50μg、100μg、150μg重組質(zhì)粒組和空質(zhì)粒組脾細(xì)胞IL-2水平均高于對照組,差異顯著(F=5.319,P0.05),150μg重組質(zhì)粒組顯著高于其他各組。免疫后第2～10周,150μg重組質(zhì)粒組脾細(xì)胞IFN-γ水平顯著高于其它各組(F=3.918,P0.05),并在第8周(A405=0.9867)時達(dá)到最高值。 結(jié)論 成功構(gòu)建了真核表達(dá)重組質(zhì)粒p3×Flag-CMW-14-TgHSP70。不同劑量重組質(zhì)粒p3×Flag-CMW-14-TgHSP70免疫小鼠均可誘導(dǎo)一定的細(xì)胞免疫應(yīng)答,150μg重組質(zhì)粒組誘導(dǎo)的脾淋巴細(xì)胞IL-2和IFN-γ水平優(yōu)于50μg、100μg重組質(zhì)粒組,50μg、100μg、150μg重組質(zhì)粒組在免疫后第8周誘導(dǎo)細(xì)胞免疫應(yīng)答達(dá)到高峰。該重組質(zhì)粒表達(dá)的抗原分子TgHSP70具免疫原性,可作為疫苗候選抗原深入研究。
[Abstract]:Objective to construct the eukaryotic expression plasmid p3 脳 Flag-CMW-14-TgHSP70 of Toxoplasma gondii RH strain. To observe the dynamic changes of cellular immune response induced by subcutaneous injection of recombinant plasmid p3 脳 Flag-CMW-14-TgHSP70. Methods: in the first part, TgHSP70 primer was designed and synthesized to amplify the TgHSP70 gene fragment. The Toxoplasma gondii TgHSP70 gene encoding fragment was obtained and ligated with eukaryotic expression plasmid p3 脳 Flag-CMW-14 after double enzyme digestion. The ligation product was identified by PCR and sequencing. The expression products of TgHSP70 in HEK293T cells were identified by RT-PCR and Western blot. Part two: 125 BALB / c mice were randomly divided into 5 groups. Mice were immunized with 50 渭 g of 100 渭 g or 150 渭 g plasmids p3 脳 Flag-CMW-14-TgHSP70 / subcutaneously for 3 times. The mice were injected with 100 渭 l Elution buffer at intervals of 2 weeks. The empty plasmid group was injected with 50 渭 g p3 脳 Flag-CMW-14, and the recombinant plasmid and empty plasmid were dissolved in 100 渭 l Elution buffer, respectively. The blood samples were collected from the eyeballs at the 2nd week and 6th week after the first immunization. The serum levels of IL-2 IFN- 緯 were determined by Elisa in 5 mice in each group. The mice were killed by dislocation of cervical vertebrae, the spleen lymphocytes were isolated and counted, the final concentration was 5 渭 g/ml and ConA5CO2 was added to culture for 24 h or 72 h at 37 鈩，

本文編號：2024021