带有GFP的小鼠Sox2基因逆转录病毒载体的构建及其检测

为了建立体细胞重编程过程中检测Sox2基因表达变化的方法,本研究利用克隆获得的小鼠Sox2基因与逆转录病毒载体pMX—IRES—GFP连接,构建带有报告基因GFP的逆转录病毒载体,将其转染293GP细胞后获得假病毒上清。将获得假病毒上清侵染小鼠成纤维细胞（NIH3T3细胞）后观察GFP的表达和检测Sox2转录量的变化。其结果,成功构建带有GFP的Sox2基因逆转录病毒载体pMX—Sox2-IRES—GFP;将构建的载体转染后293细胞后获得能侵染NIH3T3细胞的假病毒上清,且与未侵染的NIH3T3细胞相比,侵染后的NIH3T3细胞的Sox2表达量提高近10倍。本研究为开展在体细胞重编程过程中Sox2表达与重编程效率的相关性提供依据．     

论新型农村家庭经济发展的制度支持

发展与现代生产力水平和市场经济体制相适应的新型农村家庭经济具有十分重要意义。产权制度、税费制度、市场制度、合作制度、行政管理制度是推动和促进我国新型农村家庭经济发展的重要支持力量。     

渔业科学数据智能RSS阅读器的设计研究

针对传统RSS阅读器在实际应用中接收大量冗余信息的问题,提出一种智能化的RSS阅读器。该阅读器基于渔业科学数据平台,采用向量空间模型,运用中文分词、对象持久化等技术实现智能原理,设计实现了智能化RSS阅读器。实验证明：该阅读器的过滤有效性为86.2%,过滤准确性为82.4%,能够较好地过滤掉与用户不相关的信息。应用结果表明：渔业科学数据智能RSS阅读器的实现可使用户获得更精准的信息。     

家禽的转基因技术研究

转基因技术的研究虽然备受争议,但是由于具有广阔的应用前景,近年来发展十分迅速。与哺乳动物相比,禽类具有特殊的生殖系统,因此家禽的转基因研究相对较为滞后。影响转基因家禽研制的关键因素在于表达载体的选择和外源基因的导入方式,这决定了如何获得较高的孵化率和转染率。笔者对目前用于转基因家禽制备的常用载体、转基因家禽制备的方法以及转基因鸡的后续孵化技术进行了综述。     

木薯金属硫蛋白基因的克隆和表达分析

金属硫蛋白(metallothionein,MT)分子量小,半胱氨酸含量高,具有消除离子自由基,减弱重金属毒性和减少体内活性氧积累等功能。为探究木薯金属硫蛋白基因(MeMT)的原核表达能力和对活性氧的耐受性,本研究通过木薯cDNA克隆获取MeMT并将其连接至pET-28a,在BL21(DE3)菌株中成功诱导其表达。H2O2耐受性分析发现,与空载菌株相比,表达MeMT蛋白的DE3菌株对H2O2的耐受水平较高。RT-qPCR技术检测显示,在H2O2胁迫下,3 h时木薯叶片中MeMT的表达水平明显升高,但6 h时恢复甚至略低于对照组,证明MeMT在木薯中参与ROS应答过程。该结果对研究木薯抗逆性和产量的提高具有重要意义。     

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine,expressing the outer membrane protein OMP19 of Brucella species

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.     

牛乳铁蛋白基因N-lobe的cDNA克隆及毕赤酵母表达载体的构建

以牛乳腺组织总RAN为模板,经RT-PCR得到牛乳铁蛋白基因的N-lobe片段并将其克隆到pGM-T克隆载体上,最后将其连入表达载体pPICZαA中,构建牛乳铁蛋白基因N-lobe的酵母表达载体。结果表明：克隆片段大小为1028bp,与Gen Bank中登录的序列的相应部位相比,所获牛乳铁蛋白N-lobe cDNA序列的同源性为99.7%;重组表达质粒经酶切和PCR鉴定构建正确,为酵母表达乳铁蛋白基因N-lobe奠定了基础。     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

基于高光谱图像技术的小麦种子分类识别研究

为了探讨高光谱图像技术在小麦种子分类识别中应用的可行性,采集了河南地区主要种植的7个小麦品种的种子高光谱图像及900～1 700nm范围的光谱信息,建立了主成分分析法(PCA)-支持向量机(SVM)分类模型。运用PCA对光谱数据进行降维处理,结合SVM模型比较了不同实验条件下小麦种子分类准确率以及在最佳条件下3个、4个和6个品种种子的分类准确率。结果显示,3个品种间种子分类准确率除个别外平均达到95%以上,4个品种间种子分类准确率在80%左右,6个品种间种子分类准确率在66%左右。这说明充分利用光谱信息可以对3个或4个小麦品种进行多籽粒分类。     

农机维修网点急需扶持

随着国家连续几年农机购置补贴政策的实施,农机具保有量增加很快。指出在依法加强农机维修行业监督管理的同时,应注重扶持农机维修网点发展壮大,提升农机维修网点的承修能力。因此,迫切需要对有条件的农机维修网点进行政策扶持。