Expression of the human era cDNA in E.coli

Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.

A Summary of the Seminar on “Construction of China's Human Rights Discourse System in the New Era”

A seminar on Construction of China’s human Rights discourse System in the new era was held in Central South University(CSU) on April 12, 2018. The event was sponsored by the China Society for human Rights Studies and the Publicity department of the hunan Provincial Committee of the Communist Party of China, and hosted by the CSU Research Center for human Rights and the CSU School of law. nearly 70 experts and scholars in law, international relations, and communication from institutes of higher learning, research institutions, and practical work departments from around the country shared their opinions on three topics: Cultivation of a Chinese human Rights discourse System in the new era, Communication of the Chinese human Rights discourse in the new era, and Chinese human Rights discourse and International human Rights Governance in the new era. They conducted an in-depth discussion on enhancing human rights self-confidence, grasping the characteristics of the new era and the general development trend, consolidating consensus between domestic and overseas human rights discourses, and effectively carrying out international human rights exchanges. Fully reflecting the in-depth thinking and insights of attending experts and scholars on the matter, the seminar effectively promoted the development of research on Construction of China’s human rights discourse system in the new era.

The Cause of Human Rights in China Has Entered a New Era

The 19;National Congress of the Communist Party of China was an epoch-making Party Congress.The Congress solemnly declared that"socialism with Chinese characteristics has entered a new era".With the major change in China’s historical position,the cause of human rights in China has also entered a new era of socialism with

The Guiding Principles of the Cause of Human Rights in China in a New Era

The report of the 19;Communist Party of China National Congress is a document with an important historical position and unique historic contribution in the history of the People’s Republic of China and the great rejuvenation of the Chinese nation.The report can be roughly divided into two parts:general principles and substantial content.The general principles comprise four issues,which are judgment of

The Historical Position and Value Dimensions of Human Rights Civilization

Human beings are the mainstay and the ultimate goal of civilization.The history of human civilization is a continuous struggle to realize the respect,liberation,protection,and development of humanity.Human rights are an achievement of humanity and a symbol of progress,and the human rights civilization is an important component of human civilization.Understanding and interpreting human rights from the perspective of human rights civilization means that human rights are not only a concept or an idea but also a grand historical and long-term social practice.Up to now,the development of human rights civilization has roughly experienced four awakening eras:initialization,revolution,popularization,and globalization.In terms of its value dimensions,it has the characteristics of progressiveness,diversity,commonality,inclusiveness,indivisibility,openness,and so on.The historical position of human rights civilization and the development of its value dimensions have shown to the world that human rights are the common wealth of humanity,and human rights belong to all mankind;human rights are historical,concrete,and developmental;the concept of human rights is constantly evolving,and its connotations and categories are constantly expanding;achieving the free and well-rounded development of every person is the highest value realm of human rights civilization.The Chinese modernization endows Chinese civilization with modern strength and opens up new horizons for human rights civilization.The new pattern of human rights civilization to be created by Chinese modernization not only possesses the common characteristics of human rights civilization but also enjoys Chinese characteristics based on its own national conditions,enriching and developing the diversity of human rights civilization for all mankind.

兔抗人ERA抗血清的制备

目的在大肠杆菌中表达人ＥＲＡ蛋白（ｈ－ＥＲＡ）和ｈ－ＥＲＡ Ｃ端结构域蛋白，并制备兔抗ｈ－ＥＲＡ抗血清。方法以 ＰＣＲ扩增人ｅｒａ基因（ｈ－ｅｒａ）全长ｃＤＮＡ和 ｈ－ＥＲＡ Ｃ端的结构域基因，并分别克隆到（Ｈｉｓ）６融合表达载体ＰＲＳＥＴ－Ｃ和非融合表达载体ｐＤＨ中，诱导表达（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白和ｈ－ＥＲＡ Ｃ端结构域蛋白。用 ｈ－ＥＲＡ Ｃ端结构域蛋白免疫兔，制备兔抗ｈ－ＥＲＡ抗血清，并以Ｗｅｓｔｅｒｎ－ｂｌｏｔ对免抗ｈ－ＥＲＡ抗血清进行鉴定。结果大肠杆菌中表达的ｈ－ＥＲＡ Ｃ端结构域蛋白和（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白，分别占菌体总蛋白的４０％和８０％。用兔抗血清可检出大肠杆茵中表达的（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白。结论 ｈ－ＥＲＡ和 ｈ－ＥＲＡ Ｃ端结构域蛋白在大肠杆菌中获得高效表达，并成功地制备了兔抗ｈ－ＥＲＡ抗血清。

兔抗人ERA多克隆抗体的纯化

目的纯化兔抗人ERA的多克隆抗体。方法在大肠杆菌中,表达重组MBPhEra融合蛋白。表达产物先继以直链淀粉树脂亲和柱和Superose12凝胶柱过滤纯化。将纯化的MBPhEra偶联于NHSactivatedSepharoseTM上,制备亲和层析柱,纯化兔抗人ERA多克隆抗体。结果①表达、纯化的MBPhEra,相对分子质量Mr为78×103,其纯度为95%。②从抗血清中纯化获得可与MBPhEra特异性结合的兔抗hEra多克隆抗体。结论获得了特异性较好的纯化兔抗hEra多克隆抗体。

人组织era基因mRNA的表达谱

目的 研究人 era基因 m RNA在不同组织、不同细胞系的表达水平 .方法 用 α([32 p]d CTP标记人 era编码区基因作为探针 ,分别用 Northern杂交和斑点杂交分析含 12种不同成人组织 m RNA的 Multiple tissue northern(MTN)blot膜和含 76种成人、胎儿及常用细胞系 m RNA的 Multipletissue expression (MTE) array膜 ,并用同位素扫描系统对杂交结果进行图像分析 .结果 Northern blot杂交所检测的 12种组织中均有位于 2 .2 kb处的杂交条带 ,斑点杂交显示人era m RNA在所检测的 76种组织中均有表达 ,但表达水平有很大差异 ,在神经系统、某些胎儿组织以及某些肿瘤细胞中高表达 .结论 人 era m RNA表达于所有检测的组织或细胞系 ,可能为一种组成性表达的基因 .

人Era蛋白在大肠杆菌中的高效表达及其纯化

目的 在大肠杆菌中对人的 era基因 (简称 hera)进行表达、纯化 .方法 PCR扩增 hera基因 ,测序正确后克隆入大肠杆菌融合表达载体 p RSET- C,重组质粒 p RSETC-hera以大肠杆菌 BL2 1(DE3)为宿主菌 ,用 IPTG进行诱导表达 .然后利用亲和层析的方法对表达蛋白进行纯化 .结果 克隆了 hera基因 ,测序正确 ;利用 p RSET- C载体在大肠杆菌中融合表达了全长 hera- c DNA基因 ,表达量占细菌总蛋白的 5 9.6 % .融合蛋白在菌体内主要以包涵体形式存在 ,在变性条件下用 Ni- NTA亲和色谱柱纯化此融合蛋白 ,其纯度达到了 98.0 % .结论 利用基因重组技术在大肠杆菌中高表达了 hera基因 .

人era基因的克隆测序及表达

目的 克隆人的 era基因 (简称 Hera)并利用大肠杆菌进行表达 .方法 PCR扩增 Hera基因 ,测序正确后克隆入大肠杆菌融合表达载体 p GEX- 4T3,受控于 Ptac启动子 ,重组质粒 p GEX- Hera以大肠杆菌 DH5α为宿主菌 ,用 IPTG进行诱导表达 .结果 克隆了 Hera基因 ,测序正确 ;含重组质粒p GEX- Hera的菌体诱导后在 SDS- PAGE上出现一条新生蛋白带 ,相对分子质量为 6 5 ku,占菌体总蛋白的 2 3% .结论 成功扩增、克隆 Hera基因 。

人era的结构特点分析及其定点突变体的构建

目的 构建最近克隆的人era基因的定点突变体。方法 利用数据库对era的结构特点进行分析 ,在此基础上用改良的重叠延伸法分别构建人EraN端和C端的定点突变体。结果 获得了分别针对人EraN端TGP结合结构域 (位于2 9 - 36氨基酸残基 )和C端具有RNA结合活性的KH结构域 (位于 2 97- 340氨基酸残基 )的定点突变体。

重组人Era蛋白的可溶性表达、纯化及生物学活性测定

目的:为了得到可溶性表达的人Era(hEra)蛋白,并检测其生物学活性。方法:把人era的cDNA基因从pUC19质粒亚克隆入表达质粒pMAL-p2x中。pMAL-hEra转化大肠杆菌TB1,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。结果:pMAL-hEra载体表达的融合蛋白是以可溶状态存在,表达量占菌体总蛋白的23.9%。再利用直链淀粉亲和色谱柱对表达的融合蛋白进行纯化,接着用因子X将融合部分切除,但切除后的hEra蛋白不稳定,随着时间的延长而被降解。活性测定结果表明人Era蛋白能与GTP结合,并具有GTP酶的活性。结论:可溶性表达了人Era蛋白,并用体外实验证实人Era蛋白是一种G蛋白,这对人era基因的功能研究具有重要意义。

候选hEra结合蛋白A19/GST的融合表达及其抗体的制备

目的:在大肠杆菌中表达候选人Era结合蛋白A19/GST融合蛋白,并制备兔抗A19抗体。方法:利用人Era蛋白全长作为诱饵,进行胎肝cDNA文库的酵母双杂交筛选。将得到的候选人Era结合蛋白A19的编码基因序列克隆入pACT2载体中,构建pACT2A19。用PCR扩增A19蛋白的编码基因序列,克隆入融合表达载体pGEX4T3中,构建A19的表达菌,并经IPTG诱导表达A19蛋白。用SDSPAGE分析及薄层扫描检测目的蛋白的含量。用A19蛋白免疫家兔制备抗血清,以Westernblot鉴定抗体的特异性并检测抗体的效价。结果:大肠杆菌中表达的A19蛋白占菌体总蛋白的30.2%。Westernblot分析显示,抗血清具有较高的特异性,其效价约为1∶4000。结论:成功地获得了候选的人Era结合蛋白A19,制备了兔抗A19抗血清,为后续Era功能的研究奠定了基础。

6His-hEra融合蛋白的高效表达

目的 :在原核表达系统中对人Era基因进行表达。方法 :人era编码区基因克隆入pUC19质粒中 ,经全自动序列分析仪测序正确后 ,再克隆入表达载体pRSETB中 ,构建成融合 6个组氨酸的高效表达载体pRSETB Era ,转化大肠杆菌BL2 1DE3(plysS)诱导表达。结果 :经IPTG诱导后 4h ,SDS PAGE及凝胶密度扫描分析 ,表达出分子量约 4 1kD的蛋白 ,占菌体总蛋白的 5 1 2 %。结论 :该基因在大肠杆菌中的高效表达为人era的功能研究打下了基础。

Overview of the Symposium on Human Rights Cause in China in the New Era

The symposium on the "Progress of Human Rights Development in the New Era in China" was jointly hosted by the China Society for Human Rights Studies and the Publicity Department of the Guangdong Provincial Central Committee of the Communist Party of China and organized by Guangzhou University Institute for Human Rights in Guangzhou on 19 December 2017. Scholars and experts from more than 20 universities and institutes, as well as delegates from over 10 ministries and commissions under the State Council participated in the symposium. The symposium explored four themes: "Human Rights Connotations in Xi Jinping Thought on Socialism with Chinese Characteristics for a New Era", "The Progress of Human Rights Development in China after the 18 th National Congress of the CPC", "The Prospective of Human Rights Development in the New Era in China", as well as "Building a Community with a Shared Future for Human Beings and the Progress of the Human Rights Development of the World". This Paper is a record of the important opinions articulated during the Symposium.

成纤维细胞中hEra-EGFP融合蛋白的表达定位

目的 研究人 era基因在体外培养细胞中的定位 .方法 将人 era基因克隆入带绿色荧光蛋白 (EGFP)荧光标签的真核表达载体 p SMEGFP,使其位于 EGFP的 N端 ,转染NIH3T3细胞 ,于转染后 2 4和 36 h分别用荧光显微镜观察 .结果 成功构建了 EGFP- h Era融合蛋白的表达载体 ,转染细胞后可见融合蛋白位于细胞的胞质近核膜处 .结论 用EGFP融合蛋白的方法初步将 h

人Era不同结构域在酵母双杂交系统中的表达及对报告基因激活作用的检测

To construct the bait vector with different domains of human Era in yeast two-hybrid system, then assay whether its expression product can affect the growth of yeast cells and activate the reporter genes, the cDNA fragments encoding different domains of human Era was amplified by PCR, and subsequently they were cloned into pUC19. After being verified by sequencing, they were subcloned into the bait vector pGBKT7 of yeast two-hybrid system. Furthermore, these recombinant plasmids were transferred into AH109, and their expression products were assayed whether they could activate the reporter genes. The cDNA fragments encoding different domains of human Era were amplified successfully. The different domains of human Era were not toxic to AH109 and could not activate the reporter genes. Yeast two-hybrid GAL4 system could be used to fish human Era interacting protein.

人Era和人EraC端蛋白在大肠杆菌中的高表达

目的 在大肠杆菌中的高表达人 Era和人 Era C端蛋白 .方法 PCR扩增人 era基因 (h- era)的全长 c DNA和 h-era c DNA的 C端区域基因 . h- era c DNA克隆到 (His) 6融合表达载体 p RSET- C中 ,构建融合表达质粒并转化大肠杆菌BL 2 1(DE3) ,经 IPTG诱导表达 (His) 6 - h- Era融合蛋白 ;h- erac DNA的 C端区域基因克隆到非融合表达载体 p DH中 PL 启动子下游 ,转化大肠杆菌 TAP10 6 ,42℃热诱导表达人 Era C端蛋白 (h- Era- C) . SDS- PAGE电泳、凝胶薄层扫描检测蛋白的表达 .结果 表达的 (His) 6 - h- Era融合蛋白产物占全菌总蛋白的 80 % ;人 Era C端蛋白占全菌总蛋白的 40 % .结论 人 Era蛋白和 Era C端蛋白在大肠杆菌中获得了高表达 .

微媒体时代图书馆开展Human Library微阅读推广服务的研究与思考

微媒体时代不仅改变了人们的阅读方式,同时也改变了公共图书馆Human Library的阅读推广服务。通过研究微媒体时代公共图书馆开展Human Library微阅读推广的优势,进一步分析了微媒体时代公共图书馆开展Human Library微阅读推广的现状,最后着重提出笔者的几点思考,希望为公共图书馆的Human Library微阅读推广服务注入新的活力与动力。