【作者】 陈晓；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 生药学， 2004， 硕士

【摘要】 白细胞介素-10(Interleukin-10，IL-10)是一种主要由Th2细胞、巨噬细胞和活化的B细胞分泌产生的细胞因子，在免疫调节、造血及炎症过程中起重要的作用。研究发现IL-10在自身免疫性疾病、肿瘤、感染性疾病等发病中起一定调节作用外，在器官移植的排斥反应和免疫耐受中也起着相当重要的调节作用。在哮喘疾病的研究中，人们对炎性因子例如IL-2、IL-4、IL-5研究较多，而对具有抑制炎症作用的细胞因子例如IL-10研究较少。最近研究发现，IL-10通过抑制多种细胞因子的合成，对哮喘的治疗、转归具有重要意义。 目的：本文利用现代分子生物学实验方法，构建mIL-10重组腺病毒载体，制备具有活性的重组腺病毒，并对表达的mIL-10的活性进行分析检测。为研究IL-10分子生物学及药理学活性奠定了良好的基础，并为细胞因子补充和添加疗法治疗哮喘和基因治疗哮喘等临床研究做好了前期准备。 方法：从刺激后的小鼠脾细胞中克隆出mIL-10片段，利用pShuttle载体将mIL-10片段连入腺病毒载体，并用PCR反应和酶切反应对构建的重组腺病毒载体进行鉴定。分别从磷酸钙转染的pH值、DNA纯度、转染后培养时间和甘油休克时间等方面优化转染条件，根据最优条件，将mIL-10重组腺病毒载体转染入HEK 293T细胞，制备mIL-10重组腺病毒。分别用PCR反应验证病毒的制备和终点稀释检测法测定病毒的滴度。对mIL-10的表达量和活性，采用ELISA法进行定量和检测。 结果：(1)经测序证实从小鼠脾细胞中克隆的mIL-10 cDNA序列与基因库报告的序列完全一致；mIL-10片段连入腺病毒载体后经过腺病毒载体上特异引物进行PCR反应，可见312bp目的带，证实腺病毒载体构建成功；同时用Xho Ⅰ单酶切后，可见在没有重组的腺病毒载体中出现的特异条带，在阳性的重组腺病毒载体中消失，用P工一交el/工一ceu工双酶切后，可见重组腺病毒载体被双酶切为约1.skb和大于23kb的条带，单酶切和双酶切鉴定均为阳性，证实腺病毒载体构建成功; (2)经过优化磷酸钙转染法的条件，确定pH值在6.96时和纯化的质粒转染效果最好，细胞存活率高;转染后培养6h进行甘油休克效果最佳，比培养Oh直接进行休克转染效率升高584.4%;甘油休克时间为4.5 min时转染效果最佳，比不用甘油休克转染效率升高130.8%，对于HEK 293T细胞磷酸钙转染效率为8.9x 10一3一2.0x10一，; (3)病毒扩增后，有明显的CPE现象，经过腺病毒载体上特异引物进行PCR反应，可见312bP目的带，证明病毒扩增成功;终点稀释检测法测定病毒滴度为2 .5 x 1 O7pfu/ml;(4)活性分析表明，重组腺病毒在HEK 293T细胞中产生活性mIL一10。 结论:本文成功构建了mIL一10重组腺病毒载体，并通过对磷酸钙转染条件的优化，高效地将重组腺病毒载体转入HEK 293T细胞，并且成功表达了mIL一10细胞因子，得到了具有活性的mIL一10重组腺病毒，为下一步对哮喘等疾病的基因治疗奠定了坚实的基础。更多还原

【Abstract】 BACKGROUND Interleukin 10 (IL-10) is a multifunctional cytokine produced by T cells and other cell types. It regulates activities of a variety of the cells of the immune system and may play a crucial role in cross-regulation of different types of T cells. It has been reported that IL-10 has many pharmacological activities against many diseases such as self-immune, cancer and severe infections. Several inflammation cytokines are now in development for the treatment of asthma and other allergic diseases such as Interleukin-2, Interleukin-4 and Interleukin-5, but Interleukin-10 which has many special suppression functions is comparatively little considered in the treatment of asthma diseases.AIM To obtain a recombinant adenovirus which can express active mIL-10, for the further use in the treatment of asthma and other allergic diseases in gene therapy.METHODS Clone mIL-10 fragment from stimulated mouse spleen cells. Legate mIL-10 to adenovirus vector with the help of pShuttle plasmid, and detect the positive clone by PCR and restriction-enzyme digestions. Evaluate the influences of the pH value, the purity of the plasmid DNA, the cultured time before shocking and the glycerol-shock time to the calcium phosphate transfection efficiency, then transfect HEK 293T cells by optimal calcium phosphate method by using mIL-10 vector. Prepare mIL-10 recombinant adenovirus by multiple infections, then detect the virus stock by PCR and determine the virus titer by End-Point’ Dilution Assay.Determine the concentration and activity of expressed mIL-10 by ELISA.RESULTS (1) Sequencing proves that mll-10 cloned from mouse spleen cells is identical to the sequence in the GeneBank. PCR using specific primers derived from adenovirus vector results a right band at 312bp, and both Xho I digestion and Pl-Sce l/l-Ceu I double digestion can also prove a successful construction of mIL-10 recombinant adenovirus vector.(2) The results show that, the best cultured time before shocking is 6 hours, which makes the transfection efficiency increase by 584. 4% and the best time of glycerol-shock is 4. 5 minutes, making the efficiency increase by 130. 8% compared to the negative group. The pH value, of 6. 96 and the pure plasmid DNA are also a key factor, affecting the efficiency of transfection significantly. The transfection efficiency to the HEK 293T cells ranges from 8.91CT3 to 2. O10"2.(3) CPE is evident after infecting cells several times, and a right band at 312bp can be observed after PCR, all of which prove a successful preparation of recombinant adenovirus stock. The virus titer is 2. 5X 107pfu/ml determining by End-Point Dilution Assay.(4) ELISA shows the recombinant virus produces active mIL-10 in 293 cells.CONCLUSION A mIL-10 recombinant virus vector ’ is constructed successfully. It transfects HEK 293T cells in high efficiency by optimizing several influences to calcium phosphate transfection, and expressed mIL-10 protein with activity is detected then which reveals a triumphant preparation of recombinant adenovirus. The preparation of mIL-10 recombinant adenovirus establishes a good foundation for the further use in asthma animal models and the treatment for other allergic diseases in gene therapy.更多还原