基因工程的下游技术

基因工程的下游技术是现代生物技术的关键技术，它的重要性已越来越为人们所重视。生物技术产品的下游处理技术包括基因工程产物的分离、纯化及分析鉴定。本综述了基因工程产物的分离、纯化及鉴定的具体方法及鉴定标准。     

脂多糖结合蛋白的分离、纯化及其多抗的制备

目的 分离纯化大鼠脂多糖结合蛋白（LBP），并制备兔抗大鼠LBP多抗。方法 大鼠血清先通过硫酸铵盐析，再依次以Bio-Rex70阳离子交换层析和MonoQ阴离子交换层析进行纯化，纯化产物采用SDS－PAGE鉴定。用提纯的LBP免疫制备兔抗大鼠LBP的抗血清，用饱和硫酸铵盐析纯化后，经Westernblot鉴定其纯度。结果 分离纯化到较高纯度的LBP。用纯化到LBP免疫制备的兔抗LBP多抗与LBP具有良好的结合活性。结论 分离纯化到高纯度的LBP并制备出兔抗大鼠LBP多抗。     

小分子肽制备抗体方法的研究进展

许多生理调节所必须的因子都是很小的肽类, 生物技术的发展也促进了大量小分子基因工程肽类药物的出现, 因而小分子肽的分离、纯化与鉴定技术日趋重要.小分子肽的界限划分迄今未有明确定义, 一般认为小分子肽的相对分子质量(Mr)均在3000以下[1].在小分子肽的生物学功能、鉴定与其相互作用的蛋白质的研究中, 特异性抗体是一重要的研究工具[2].鉴于小分子肽仅有反应原性, 免疫原性较弱,因此在小分子肽制备抗血清过程中采取多种方法.     

单克隆抗体亲和层析一步纯化重组人生长激素

单克隆抗体亲和层析技术可用来从体液、组织抽提液、细胞培养液中,特别是从基因工程菌体破碎液等复杂组成物中分离出微量蛋白质和多肽.由于其特异性高,操作简便而迅速,在高价值蛋白质和治疗用多肽的下游处理过程中,作为一种良好的分离纯化工具,有较广泛的适用性.本文运用该技术从人生长激素(hGH)基因工程菌发酵破碎液中一步分离制备高纯度并具有生物活性的重组hGH.     

层粘连蛋白特异性受体--整合素α6亚单位胞外区单克隆抗体的制备及鉴定

目的：制备针对整合素α6胞外区的单克隆抗体并鉴定其生物学特异性。方法：利用整合素α6 cDNA构建可表达整合素α6胞外区的原核表达载体，在大肠杆菌中表达GST－整合素α6胞外区融合蛋白。融合蛋白经凝胶分离纯化后免疫Balb/C小鼠，取其脾细胞，用传统杂交瘤技术与小鼠骨髓瘤细胞融合及次黄嘌呤，氨基喋呤与胸苷选择性培养。单克隆杂交瘤上清经ELISA双筛后，分析其亚类及进行免疫细胞化学鉴定。结果：经ELISA双筛，获得了一株能稳定分泌对整合素α6胞外区的单克隆抗体的杂交瘤细胞株，其分泌抗体亚类为IgG1。免疫细胞化学显示此单抗在人肝癌细胞系BEL－7402呈强阳性反应。结论：通过原核表达产物制备并纯化了针对整合素α6胞外区的单克隆抗体，对分离纯化α6亚单位、研究其生物学作用及鉴定正常和疾病状态下是否表达整合素α6及其水平具有重要意义。     

天花粉蛋白突变体TCSFYY163-165CSA的构建表达与纯化

目的:构建天花粉蛋白(TCS)突变体基因,并在原核系统进行表达及纯化。方法:应用计算机预测TCS分子上可能的抗原决定簇(FYY163-165),并进行定点突变。以栝楼基因组DNA为模扳,经PCR扩增突变基因,与pRSET-A表达载体连接,转化感受态E.coli DH5α,提取质粒进行酶切鉴定及测序。将阳性重组质粒转化感受态E.coli BL21(DE3),经IPTG诱导表达后,对表达产物进行Western blot法鉴定和Ni-NTA层析纯化。结果:构建了带有TCS突变体基因(TCSFYY163-165CSA)的重组表达质粒,使目的蛋白在大肠杆菌中获得高效可溶性表达,表达产物经纯化后,得到均一的TCS突变体蛋白。结论:成功地构建了TCS突变体基因TCSFYY163-165CSA,并获得高效表达,为基因工程方法改造TCS提供了新的途径。     

重组人血红蛋白δ链的制备与鉴定

目的克隆人血红蛋白δ（delta）链蛋白基因，并在大肠杆菌中进行表达，经纯化与鉴定，获得δ链蛋白，为建立β-地中海贫血的免疫学筛查方法提供特异性抗原。方法从K562细胞中提取总RNA，采用RT-PCR技术获得目的片段并将其克隆到pET-32a及pET43．1a载体中，经PCR、酶切及测序验证，以IPTG诱导其在大肠杆菌中表达。包涵体经变性、复性后以Ni^2＋亲和层析柱纯化，可溶性蛋白直接以Ni^2＋亲和层析柱纯化。采用Western Blot和ELISA分析鉴定表达产物。结果成功构建两种融合表达载体pET32-Hbδ及pET43．1-Hbδ，分别为包涵体表达和可溶性表达，纯化产物经Western blotting和ELISA鉴定均为δ链蛋白。结论克隆表达并获得了纯化的人血红蛋白δ链，为后续的抗体制备及地中海贫血的免疫筛选方法的建立提供了抗原。     

人源受体相关蛋白的原核表达及功能检测

目的研究人源受体相关蛋白(RAP)的表达并鉴定表达产物的功能。方法用IPTG诱导培养含有人源RAP重组表达载体(pET-28 a( )-RAP)的BL21转化菌,利用融合蛋白中6个组氨酸纯化标签,经N i -NTA树脂亲和层析、SDS-PAGE分离鉴定表达产物,并经脱盐、浓缩、冻干处理获得RAP融合蛋白冻干品。将RAP融合蛋白与富含LDLR家族受体的巨噬细胞进行结合实验,分析RAP融合蛋白的功能。结果经亲和层析分离纯化后获得可溶性表达的RAP融合蛋白。实验显示,表达的RAP融合蛋白可竞争性抑制VLDL与巨噬细胞的结合。结论实验获得的RAP融合蛋白具有正常的结合活性,可用于相关研究,为RAP蛋白生产开发奠定了良好的基础。     

离体脊髓星形胶质细胞的纯化与鉴定

目的探索新生大鼠脊髓星形胶质细胞的分离、培养与传代及纯化方法，鉴定其培养纯度。方法分离、培养新生大鼠脊髓星形胶质细胞，用换液时暴露结合旋转法加以纯化，酶消化法传代后进行免疫组化鉴定，并染核计数星形胶质细胞的纯度。结果星形胶质细胞贴壁快，多在12h内，3d后数量显著增加；免疫细胞化学染色显示，NF200抗体染色为阴性，提示培养盖片中不含神经元；GFAP阳性染色细胞高达95．8％。结论换液时短暂暴露结合恒温旋转法可纯化得到高纯度的星形胶质细胞，是一种简单、高效的星形胶质细胞纯化方法。     

Luffin P1-KDEL的构建、表达、纯化及鉴定

目的构建植物毒素Luffin P1的原核表达质粒PET32a(+)-Luffin P1,并对其进行诱导表达,以及表达蛋白的纯化及鉴定。方法采用基因工程技术将重组基因片段Luffin P1克隆到表达载体pET32a(+)中,经酶切及DNA序列分析正确后,转化至表达宿主菌BL21(DE3)中,经IPTG诱导表达Luffin P1融合蛋白,并用His标签抗体对该融合蛋白进行Western blot鉴定,对鉴定正确的融合蛋白进行纯化,肠激酶酶切及再纯化。结果成功的构建了原核表达载体pET32a(+)-Luffin P1,获得了纯度较高的Luffin P1蛋白。结论通过基因工程合成Luffin P1蛋白是成功的,并为进一步对Luffin P1功能研究及制备免疫毒素的弹头鉴定了实验基础。     

Genetic engineering of allergens: future therapeutic products

Genetic engineering of allergens for specific immunotherapy should aim at the production of modified molecules with reduced IgE-binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reactive with the natural allergen (blocking antibodies). Common approaches for engineering of hypoallergens usually require knowledge of T and B cell epitopes and involve changing specific base pairs (mutated gene), introduction of a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), and deletions (truncated gene or fragments). DNA family shuffling has the advantage that it does not require a priori knowledge of structural and functional properties for efficient generation of hypoallergens. The combination of the hypoallergen concept with the Th1-inducing genetic immunization approach might be an attractive alternative for protein-based immunotherapy.     

PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.     

三峡库区上、下游血吸虫病流行区钉螺线粒体cox1基因遗传变异研究

目的研究三峡库区上、下游血吸虫病流行区钉螺线粒体DNA细胞色素C氧化酶亚单位t（c毗1）基因的遗传变异。方法采集三峡库区上游四川、云南及下游安徽、湖北4省共7个地、市的钉螺样本,提取基因组DNA,PCR特异性扩增线粒体cox1基因并测序,用ClustalX（1．81）软件进行多序列比对,MEGA（4．0）软件Kimura2-parameter法计算遗传距离,邻接法（NJ）和最大简约法（MP）构建系统发生树。结果上游与下游不同地域株钉螺间cox1基因差异约为16％．下游地区的肋壳与光壳钉螺cox1基因差异约为3．7％,上游不同地域株钉螺碱基差异约为5．4％。遗传距离显示,上游四川与云南地域株的遗传距离为0．022～0．050,下游安徽与湖北地域株的遗传距离为0．014～0．027,而上游与下游各地区螺群间的遗传距离在0．127～0．138之间,明显大于上游或下游各地区螺群内的遗传距离。进化树结果表明,下游湖北的荆州、石首和安徽的芜湖、宁国钉螺形成一支系,上游四川的绵竹、新都钉螺同属一支系。但两种方法构建的进化树在云南大理钉螺的归属上存在差异,MP法提示大理钉螺从上游的分支中独立出来,单独形成一类。结论上游与下游不同地域株钉螺cox1基因遗传差异较显著,下游肋壳和光壳钉螺种群内遗传变异较小,而上游光壳钉螺种群内遗传变异较大。     

共表达HIV-1 gag-gp120与白细胞介素6重组鸡痘病毒的筛选

目的构建共表达中国株HIV1gaggp120与白细胞介素6(IL6)的重组鸡痘病毒(FPV)。方法分别将HIV1gaggp120基因和IL6基因插入到FPV表达质粒pUTAL复合启动子和单一启动子下游,构建重组FPV表达质粒pUTAGEIL6。利用脂质体法将重组质粒和FPV282E4株共转染鸡胚成纤维细胞,经BUdR加压筛选,重组病毒分别用SDSPAGE和WesternBolt进行鉴定,观察病毒样粒子的形成和重组病毒在哺乳动物细胞中的表达,并分析其免疫原性。结果阳性重组病毒基因组点膜处有显色斑点,WesternBolt结果显示重组病毒表达了gaggp120嵌合蛋白和IL6,在病毒感染细胞中有反转录病毒样粒子形成,且重组病毒可在哺乳动物细胞中表达目的蛋白。小鼠免疫指标检测表明该重组病毒具有很好的免疫原性。结论成功构建了共表达中国株HIV1gaggp120与IL6的重组FPV,为HIV-1基因工程活载体疫苗和巨分子颗粒化疫苗的制备奠定了基础。     

Application of the adverse outcome pathway framework to genotoxic modes of action

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114–134, 2020. © 2019 Wiley Periodicals, Inc.     

The role of the T-cell costimulatory molecule Tim-1 in the immune response

Upon encountering antigen, CD4+ T-cells become activated and can differentiate into subsets with distinct functional characteristics. One of these subsets is the Th2 cell, which generates large amounts of interleukin (IL)-4, -5, -10 and -13 in response to a subsequent encounter with antigen. In the context of a protective immune response, Th2 cells promote immune cell activation, antibody production, and inflammatory responses that help clear infections. However, aberrant responses by Th2 cells can lead to debilitating allergic diseases such as asthma. Thus, a reasonable approach toward gaining novel insights into immunity and allergic disease is to define the mechanisms that control Th2 cell differentiation and mature Th2 cell function. Recent work suggests that a protein called Tim-1 (T-cell immunoglobulin and mucin protein-1) is expressed on CD4+ T-cells and plays a central role in regulating Th2 responses. Genetic analysis has linked polymorphisms in the human TIM1 gene to susceptibility to allergic disease, while studies involving mice have shown that ligation of Tim-1 promotes CD4+ T-cell activation. The signal transduction pathways downstream of Tim-1 are a relatively unexplored area. Continued study will undoubtedly reveal novel insights regarding the relationships between Tim-1, Th2 responses, and allergic disease.     

骨质疏松的分子遗传学研究进展

遗传因素在骨质疏松的发病中起重要作用。本文简要介绍近年来在骨质疏松相关基因关联分析方面所取得的研究进展,并对新近发现的删基因在骨量维持中作用的研究进展进行了重点介绍,LRP5基因在骨量和骨密度调控中起着关键作用。     

Toll signaling pathways in the innate immune response

The Toll signaling pathway, which is required for the establishment of the dorsal-ventral axis in Drosophila embryos, plays an important role in the response of larval and adult Drosophila to microbial infections. Recent genetic evidence has shown that a mammalian Toll-like receptor, mouse Tlr4, is the signal transducing receptor activated by bacterial lipopolysaccharide. Thus, Toll-like receptors appear to detect a variety of microbial components and to trigger a defensive reaction in both Drosophila and mammals. Genetic data from both Drosophila and mice have defined components required for activation of Toll-like receptors and for the downstream pathways activated by the Toll-like receptors.     

Genetic examination in clinical laboratory

Genetic technology is finding active application today in the field of clinical laboratory medicine. Genetic examinations are divided into following three main classes: 1) examination for infectious disease according to the detection of the gene derived from bacteria or viruses, 2) examination for inherited disease according to molecular analysis of the genetic variation, 3) examination for oncogene according to molecular analysis of genetic abnormalities. At present, the main genetic examination in a large number of laboratories is for infectious disease because of its relatively simplified technique and high demand. The division of genetics is not a new independent section of clinical laboratory, but rather an ultramodern and powerful tool for existing divisions, such as biochemistry, serology, hematology, microbiology, and pathology. Genetic technology quickly provides results with high sensitivity and reliability, and plays a role at the core of the clinical laboratory. We should remember that the genetic technology is a great present given to clinical laboratories, however, it will eventually change into only one of the routine examinations according to the method of used. Examinations utilized in the clinical laboratory must be well established and standardized. Genetic examinations are no exception to that rule. These tests require a remarkably high precision since the results have an extraordinarily important meaning. There are more than 8,000 inherited diseases for instance. It is difficult to cover all examinations for those 8,000 in one laboratory. We need a network of laboratories that possess a genetic division, so that the examinations for as many inherited disease as possible can be comprehensively offered.     

Development and implementation of an electronic medical record module to track genetic testing results

PurposeGenetic testing and results return pose many challenges, even in the era of electronic medical records. Whether results are positive or negative, genetic testing and return of results necessitate patient follow-up, referrals, and coordination between providers. Genetic evaluations typically utilize a variety of testing modalities with differing timetables and/or avenues to return. Therefore, genetic information requires a secondary, unified mechanism for storing and tracking results and communication to facilitate patient care.MethodsWe developed an electronic medical record (EMR) episodes-based module called Pediatric Genetic Tracking to provide a centralized summary of patient tracking information in a single-institution pediatric genetics setting.ResultsWe created episodes for 6,133 patients evaluated in our division over a 3-year period. They highlighted clinical information for 1,901 different diagnoses and 547 genetic tests, and the involvement of 9 providers, 7 genetic counselors, 61 trainees, and 15 students using two modes of follow-up.ConclusionThis Pediatric Genetic Tracking episodes system serves as a “one-stop shop” living document for updated patient genetic information and can be easily expanded to include variant content for broader population level sharing or analysis. These episodes-based modules facilitate communication to support timely and accurate return of genetic test results and follow-up.