刚地弓形虫HSP70基因重组质粒的构建及其诱导小鼠的细胞免疫应答

发布时间：2018-06-15 23:06

  本文选题：刚地弓形虫 + 热休克蛋白70　； 参考：《山西医科大学》2011年硕士论文

【摘要】：目的 构建弓形虫RH株p3×Flag-CMW-14-TgHSP70真核表达重组质粒。观察不同剂量的重组质粒p3×Flag-CMW-14-TgHSP70皮下肌肉注射免疫BALB/c小鼠诱导的细胞免疫应答动态变化。 方法 第一部分:设计合成TgHSP70引物,PCR扩增其基因片段,获得弓形虫速殖子热休克蛋白(TgHSP70)基因编码片段,双酶切后与真核表达质粒p3×Flag-CMW-14连接,连接产物经酶切、PCR及测序鉴定。脂质体法将重组体转染HEK 293T细胞中,TgHSP70的表达产物通过RT-PCR和Western blot在基因和蛋白两个水平进行鉴定。 第二部分:125只BALB/c小鼠随机均分5组,分别用50μg、100μg、150μg重组质粒p3×Flag-CMW-14-TgHSP70/只皮下肌肉注射免疫小鼠3次,各间隔2周,空白对照组注射100μl Elution缓冲液,空质粒组注射50μg p3×Flag-CMW-14,重组质粒和空质粒分别溶于100μl Elution缓冲液中。分别于首免后第2、4、6、8、10周摘眼球采血,分离血清,每组5只,ELISA法测定血清中IL-2、IFN-γ水平。颈椎脱臼处死小鼠,无菌取脾,分离脾淋巴细胞并计数;再加入终浓度为5μg/ml的ConA,5%CO2、37℃培养24h、72h,取培养上清。ELISA法测定培养24h上清液中IL-4、IL-2水平和培养72h上清液中IFN-γ水平。 结果 PCR扩增获得约495 bp的HSP70编码基因片段,p3×Flag-CMW-14-TgHSP70重组体构建成功,阳性克隆经双酶切、PCR及测序鉴定,基因片段序列完全正确。将p3×Flag-CMW-14-TgHSP70真核表达重组质粒转染到HEK 293T细胞中,经RT-PCR检测可见预期大小的目的基因条带,Western-blot检测表达产物大小约19 kDa。 50μg、100μg、150μg重组质粒组小鼠血清IL-2水平在免疫后第6周明显上升,第8周达到顶峰,第10周稍有回落。50μg、100μg、150μg重组质粒组和对照组的血清IFN-γ水平随时间改变基本一致,无明显差异。免疫后第10周,50μg重组质粒组脾细胞数(17.362×10~6个/ml)显著高于150μg组(15.22×10~6个/ml)、100μg组(15.66×10~6个/ml)、空质粒组(14.82×10~6个/ml)和空白对照组(16.076×10~6个/ml)(F=4.478,P0.05)。免疫后第2～10周150μg组脾淋巴细胞IL-4水平均高于50μg和100μg组,但差异无统计学意义。免疫后第2～10周,50μg、100μg、150μg重组质粒组和空质粒组脾细胞IL-2水平均高于对照组,差异显著(F=5.319,P0.05),150μg重组质粒组显著高于其他各组。免疫后第2～10周,150μg重组质粒组脾细胞IFN-γ水平显著高于其它各组(F=3.918,P0.05),并在第8周(A405=0.9867)时达到最高值。 结论 成功构建了真核表达重组质粒p3×Flag-CMW-14-TgHSP70。不同剂量重组质粒p3×Flag-CMW-14-TgHSP70免疫小鼠均可诱导一定的细胞免疫应答,150μg重组质粒组诱导的脾淋巴细胞IL-2和IFN-γ水平优于50μg、100μg重组质粒组,50μg、100μg、150μg重组质粒组在免疫后第8周诱导细胞免疫应答达到高峰。该重组质粒表达的抗原分子TgHSP70具免疫原性,可作为疫苗候选抗原深入研究。
[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- 纬 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，

本文编号：2024021