缝隙连接蛋白50真核表达载体的构建与表达

目的:构建pEGFP/缝隙连接蛋白(connexin,Cx)50真核表达载体,观察其在细胞内的表达和功能情况。方法:经PCR获得Cx50基因片段,重组至有增强绿色荧光蛋白标记的真核表达载体pEGFP-N1中,经脂质体转染至宫颈癌Hela细胞中,通过荧光显微镜检测蛋白表达。结果:转染细胞有增强绿色荧光蛋白及Cx50的表达。结论:融合表达增强绿色荧光蛋白后,不影响缝隙连接蛋白的功能。     

A546D突变型TGFBI基因真核表达载体的构建及体外表达

目的 构建A546D突变型TGFBI基因真核表达载体和体外转染体系,为研究野生型及A546D突变型TGFBI基因的功能提供实验基础.方法 应用重叠延伸PCR法定点诱变,构建A546D突变型TGFBI基因真核表达载体,以阳离子脂质体介导体外转染技术瞬时转染人角膜上皮(human corneal epithelium,HCE)细胞,Western Blot法检测转染后培养基中HCE细胞分泌的TGFBI蛋白的表达.结果 重组质粒总长约7.5 kb,由HindⅢ、Xba Ⅰ双酶切鉴定产生5.4 kb和2.1 kb两条片段者为克隆正确质粒.测序鉴定证实了TGFBI基因编码区完整插入到载体pcDNA3.1的多克隆位点中,突变载体的第1685位碱基由C替换为A,使其编码的第546个氨基酸由Ala突变为Asp即A546D突变.野生型及A546D突变型载体转粢入HCE细胞后,TGFBI蛋白有效表达.结论 成功构建了A546D突变型TGFBI基因真核表达载体,为TGFBI基因相关性角膜营养不良致病机制的研究奠定了基础.     

脂质体包裹人促甲状腺激素受体胞外段基因真核表达质粒的构建

目的:构建阳离子脂质体包裹的人促甲状腺激素受体胞外段基因真核表达质粒。方法:PCR扩增穿梭质粒PHMCMVTSHR289目的基因并连接于真核表达质粒pcD NA3.1+上,重组质粒pcD NA3.1+/TSHR289采用酶切及测序法鉴定。阳离子脂质体包裹重组质粒pcD NA3.1+/TSHR289。结果:重组质粒pcD NA3.1+/TSHR289用Hind III酶切后产物经0.8%琼脂糖凝胶电泳检测显示出现512bp条带。正向测序发现AAC突变为AAT,为同义突变。反向测序发现GCG突变为GCT,亦为同义突变。阳离子脂质体与重组质粒的体积质量比例为3∶1。结论:酶切及测序鉴定重组质粒pcD NA3.1+/TSHR289构建成功。     

内皮抑素基因定点突变体的构建

目的:内皮抑素是一种内源性的血管生成抑制剂。运用定点突变技术对人内皮抑素基因进行改造,将内皮抑素中的RGAD改为RGD序列。构建野生型和突变型RGD-en-dostatin基因的真核表达载体,为进一步探讨其对角膜新生血管的影响奠定基础。方法:以pMD18-T/endostatin载体为模板扩增野生型内皮抑素基因全长编码序列,克隆至真核表达载体pIRES2-EGFP上,采用快速体外定点诱变技术获得RGD突变体。经PCR,酶切和序列测定方法鉴定重组质粒。结果:PCR成功扩增了野生型和RGD突变型的内皮抑素基因,并成功构建了其真核表达载体。结论:改造的含有RGD结构的内皮抑素基因真核表达质粒的成功构建并鉴定。     

蛋白酶体β5亚单位基因真核表达载体的构建

目的:构建蛋白酶体亚单位β5基因真核表达质粒。方法:从人晶状体上皮细胞株SRA01/04中提取总RNA,经RT-PCR扩增获得β5亚单位的全长cDNA片段,将其克隆至真核表达载体pcDNA3.1上。结果:RT-PCR法扩增出约792bp的β5亚单位基因全部编码序列的片段。酶切鉴定和测序分析证实所插入的β5亚单位的基因序列完全正确。结论:成功构建了蛋白酶体β5亚单位基因真核表达重组质粒。     

人TGFBI基因克隆及真核表达载体的构建

目的:克隆人TGFBI基因,并构建其真核表达载体.方法:通过RT-PCR从供体角膜移植术后留下的角膜组织中克隆人TGFBI基因全长编码区,将该DNA片断亚克隆到克隆载体pMD18-T中,进一步通过和SalI双酶切将目的片断定向克隆至真核表达质粒载pCI中.经PCR,酶切和序列测定方法鉴定重组质粒.结果:获得了人TGFBI的编码基因,并成功建了其真核表达载体.结论:人TGFBI基真核表达质粒的成功构建并鉴定.     

小鼠TGFBI基因真核表达载体的构建和表达

目的构建小鼠TGFBI基因的真核表达载体,为研究角膜营养不良的发病机制奠定基础。方法提取BALB/cBy小鼠正常角膜组织总RNA,经反转录-PCR合成TGFBI cDNA,克隆入真核表达载体pcDNA3.1并测序验证。用不同剂量重组质粒pcDNA3.1-TGFBI转染NIH3T3细胞,通过SYBR荧光实时定量PCR和Western blot检测TGFBI在细胞中的表达。结果测序结果显示扩增到的TGFBI cDNA以正确序列和方式插入载体,实时定量PCR和Western blot结果显示TGFBI在NIH3T3细胞中表达增强。结论成功构建了小鼠TGFBI基因真核表达载体,并在细胞中进行表达,为进一步研究TGFBI在角膜内的生理、病理功能奠定基础。     

pEGFP-N1-Endostatin重组质粒的构建及其体外表达

目的构建分泌型人内皮抑素（ES）的真核表达载体pEGFP-N1-Endostatin重组质粒,鉴定其蛋白的体外瞬时表达。方法以pcDNA3-Endo质粒为模板,通过PCR扩增获得人ES基因片段,且在基因前加信号肽序列,将其定向插入真核表达载体pEGFP-N1中,获得重组质粒pEGFP-N1-ES。采用HindⅢ和BamHⅠ双酶切法、PCR法及插入片段序列测定法鉴定该质粒。利用阳离子脂质体介导法,将其转染到人胚胎肾HEK-293细胞中,采用免疫组织化学法和Westernblot法检测转染细胞内及培养上清液中ES蛋白的表达。结果通过HindⅢ和BamHⅠ双酶切、PCR及测序鉴定证明构建出了含ES基因的真核表达载体,且插入片段正确。采用免疫组织化学法和Western blot法检测表明HEK-293细胞内及培养上清中存在相对分子质量为20 000的人ES蛋白表达。结论成功构建了重组质粒pEGFP-N1-ES真核表达载体,转染HEK-293细胞后可稳定有效地分泌人ES蛋白。     

单纯疱疹病毒I型截短糖蛋白B基因序列的克隆及鉴定

目的构建单纯疱疹病毒I型（herpes simplex virusI，HSV—I）SM44株截短糖蛋白B基因序列，为研制联合基因疫苗奠定基础，用于角膜炎的防治。方法利用聚合酶链反应（polymerase chain response，PCR）技术从感染HSV—I SM44株的vero细胞中扩增出HSV-gBt编码基因，经双酶切鉴定后将目的基因定向插入真核表达质粒pcDNA，载体中，构建出重组真核表达质粒pcDNA3-gBt，并对其进行酶切分析和测序鉴定。结果对pcDNA3-gBt双酶切后，电泳可见目的基因（1．5kb）和线性质粒pcDNA3（5．4kb）两条带；测序结果表明，克隆基因插入方向正确，与基因库中登录的HSV-1 F株gB基因序列比较，同源性达99．5％。结论经证实成功地构建了重组真核表达质粒pcDNA3-gBt，为进一步研究其免疫学效应及构建病毒糖蛋白联合疫苗并最终用于单疱病毒角膜炎的预防和治疗奠定理论基础.     

Figure 1 CD40 , CD68 & CD40L expression in the epidermis and subcutaneous tissue of erythema nodosum (×200) A: CD40+ (red) (AEC) in epidermis；B: CD68＋ (black) (BCIP/NBT) in subcutaneous tissue；C: CD40L (red) (AEC) in subcutaneous tissue Bioinformatics analysis and construction of eukaryotic expression plasmid of Cx50 V64G mutation

AIM: To construct and analyze eukaryotic expression plasmid inserted by Cx50 with V64G mutation through bioinformatics software. METHODS: The full coding domain sequence of Cx50 with V64G mutation was acquired from the blood of patients with cataract and was cloned into pcDNA3.1/Amp (+).The constructed plasmid was identified with PCR , enzyme digestion and sequencing. The analysis of Cx50 with V64G mutation was performed with bioinformatics software. RESULTS: Cx50 with V64G mutation was successfully amplified and its eukaryotic expression plasmid was constructed. Valine-64 is well conserved in the first extracellular loop of connexin 50 in different species and also in different human α-type gap junctional proteins. CONCLUSION: The successive reconstruction and verification of eukaryotic expression plasmid containing Cx50 with V64G mutation established the foundation for further studying the mechanism of cataract.     

特殊表型先天性白内障小鼠晶状体中细胞缝隙连接蛋白的表达

目的观察γD-晶状体蛋白点突变导致的小鼠先天性白内障表型,检测该特殊表型先天性白内障小鼠晶状体中细胞缝隙连接蛋白（Cx）的表达。方法观察突变小鼠出生后不同时间晶状体的形态学变化;应用免疫荧光染色法分析晶状体内Cx46和Cx50的表达和分布。结果突变小鼠模型呈稳定一致的显性遗传,出生后7 d即表现为明显的核性白内障,出生后21 d纯合子小鼠晶状体混浊严重,后囊膜自然破裂;免疫荧光染色分析发现突变小鼠晶状体内Cx46和Cx50的表达均出现下降,越靠近晶状体中心部下降越明显。结论γD-晶状体蛋白点突变可导致晶状体后囊膜破裂这种特殊表型的先天性白内障,白内障的形成和后囊膜破裂的产生与晶状体内Cx46和Cx50的表达下降有关。     

Characterization of a mouse Cx50 mutation associated with the No2 mouse cataract.

PURPOSE: Recently, a missense mutation in the mouse connexin 50 (Cx50) gene has been associated with the nuclear opacity 2 (No2) mouse cataract. This missense mutation (D47A) resulted in an aspartate-to-alanine substitution at amino acid position 47 in the first extracellular domain of Cx50. To better understand the role of Cx50 in the pathogenesis of congenital cataract, the functional consequences of the D47A mutation in the Xenopus oocyte expression system were studied. METHODS: D47A was constructed using polymerase chain reaction (PCR) mutagenesis. Xenopus oocytes were injected with in vitro transcribed cRNA encoding wild-type mouse Cx50 (Cx50wt), wild-type rat Cx46 (Cx46wt), D47A, or combinations of wild-type and mutant connexins. The oocytes were then devitellinized and paired. Gap junctional conductance (Gj) was measured using a dual two-microelectrode voltage-clamp technique. RESULTS: Homotypic oocyte pairs expressing wild-type Cx50 or Cx46 were well coupled. In contrast, oocytes injected with D47A cRNA did not form gap junctional channels when paired homotypically. To test whether the D47A mutation could interact with wild-type connexins in a dominant negative manner, oocytes were injected with equal amounts of mutant and wild-type connexin cRNA, mimicking the heterozygous condition. Expression of D47A did not inhibit the development of junctional conductance in paired oocytes induced by wild-type Cx50 or Cx46. CONCLUSIONS: These results indicate that the D47A mutation acts as a loss-of-function mutation without strong dominant inhibition. In No2 mice, the mutation would be predicted to result in a reduction in intercellular communication, leading to cataractogenesis. It may also cause other qualitative changes such as a change in permeability for small molecules.     

A novel mutation of p.F32I in GJA8 in human dominant congenital cataracts

AIM: To identify a causative mutation in a three-generation family with autosomal dominant congenital total cataract and dissect the molecular consequence of the identified mutation. METHODS: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. Mutation were screened in recruited family members by polymerase chain reaction (PCR) of the two reported genes (CRYAA and GJA8) which were linked to human total cataracts and direct sequencing of the PCR product. The molecular consequences of the identified mutation was dissected. The plasmids carrying wild-type and mutant mouse ORF of Gja8, coding for connexin 50 (Cx50), were generated and ectopic expressed in 293 cells. Recombinant protein expression and cellular localization of recombinated Cx50 were assessed by confocal microscopy. RESULTS: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. Mutation were screened in recruited family members by PCR of the two reported genes (CRYAA and GJA8) which were linked to human total cataracts and direct sequencing of the PCR product. The molecular consequences of the identified mutation was dissected. The plasmids carrying wild-type and mutant mouse ORF of Gja8, coding for Cx50, were generated and ectopic expressed in 293 cells. Recombinant protein expression and cellular localization of recombinated Cx50 were assessed by confocal microscopy. CONCLUSION: This study has identified a novel cataract mutation in GJA8, which adds a novel mutation to the existing spectrum of Cx50 mutations with cataract. The molecular consequences of p.F32I mutation in GJA8 exclude instability and the mislocalization of mutant Cx50 protein.     

Genetic linkage analyses and Cx50 mutation detection in a large multiplex Chinese family with hereditary nuclear cataract

Purpose: The aim of the study was to characterize the underlying mutation in a large multiplex Chinese family with hereditary nuclear cataract.

Methods: A 6-generation Chinese family having hereditary nuclear cataract was recruited and clinically verified. Blood DNA samples were obtained from 53 available family members. Linkage analyses were performed on the known candidate regions for hereditary cataract with 36 polymorphic microsatellite markers. To identify mutations related to cataract, a direct sequencing approach was applied to a candidate gene residing in our linkage locus.

Results: A linkage locus was identified with a maximum 2-point LOD score of 4.31 (recombination fraction?=?0) at marker D1S498 and a maximum multipoint LOD score of 5.7 between markers D1S2344 and D1S498 on chromosome 1q21.1, where the candidate gene Cx50 is located. Direct sequencing of Cx50 showed a 139 G to A transition occurred in all affected family members. This transitional mutation resulted in a replacement of aspartic acid by asparagine at residue 47 (D47N) and led to a loss-of-function of the protein.

Conclusions: The D47N mutation of Cx50 causes the hereditary nuclear cataract in this family in an autosomal dominant mode of inheritance with incomplete penetrance.     

先天性核性白内障家系致病基因的克隆及在真核细胞中的表达

目的克隆1个先天性核性白内障家系致病基因-突变型GJA8（mGJA8）,研究其编码的蛋白质在真核细胞系中的表达和定位。方法从研究发现的先天性核性白内障家系患者基因组中扩增突变型mGJA8,克隆入载体pGEF—T,然后酶切,定向克隆到真核表达质粒pEGFP-N1中,构建pEGFP—N1-mGJA8重组表达质粒,然后用限制性内切酶消化和DNA测序鉴定,最后通过脂质体包埋转染法,用pEGFP—N1-mGJA8和pEGFP—N1质粒转染COS-7细胞,荧光显微镜观察其在COS-7细胞内的表达,采用细胞免疫组织化学法验证蛋白表达。结果经双酶切和DNA序列测定,证实致病基因mGJA8重组质粒构建成功,荧光显微镜观察在COS-7细胞上致病基因mGJA8蛋白表达,细胞免疫组织化学法显示致病基因表达蛋白在COS-7细胞中特异性表达。结论成功克隆出该家系的致病基因mGJA8,并且完成了该致病基因在体外真核细胞中的特异性表达,为进一步研究该家系的发病机制奠定基础。     

Characterization of a mutation in the lens-specific MP70 encoding gene of the mouse leading to a dominant cataract

During an ethylnitrosourea mutagenesis screen, Aey5, a new mouse mutation exhibiting an autosomal dominant congenital cataract was isolated. The cataractous phenotype is visible at the eye opening and progresses to a nuclear and zonular cataract at 2 months of age with no difference in onset or severity between heterozygous and homozygous mutants. Histological analysis revealed that fiber cell differentiation continues at the lens bow region, but the cell nuclei do not degrade normally and remain in the deeper cortex. Further, the lens nucleus has clefts of various sizes while the remainder of the eye was morphologically normal. The mutation was mapped to chromosome 3 between the markers D3Mit101 and D3Mit77 near the connexin encoding genes Gja5 and Gja8. Sequence analysis revealed no differences in the Gja5 gene, but identified a T-->C mutation at position 191 in the Gja8 gene, which was confirmed by an additional Mva 12691 restriction site in the genomic DNA of homozygous mutants. This mutation results in Val-->Ala substitution at codon 64 of connexin50 (Cx50) also known as lens membrane protein 70 (MP70). Aey5 represents the second dominant mouse cataract mutant affecting Cx50, a membrane protein preferentially expressed in the lens. Since both mutations affect similar regions in the first extracellular domain this region appears to be critically important for its function in lens transparency.     

中国南方先天性白内障一家系GJA8基因C>G新突变

目的 对一个常染色体显性遗传先天性白内障（ADCC）家系进行候选基因筛查,以期寻找其可能的致病基因。设计 实验研究。研究对象 一个中国南方ADCC家系。方法 应用聚合酶链反应（PCR）和DNA直接测序方法,对该家系进行ADCC常见致病基因突变筛查。主要指标 基因序列。结果 临床眼科检查显示该家系先天性白内障类型为核性白内障。候选基因序列测定显示在GJA8基因c.565位置上存在C>G的突变,该突变导致在缝隙连接蛋白Cx50 p.189位置上的脯氨酸突变为丙氨酸,此氨基酸改变位点位于缝隙连接蛋白结构中第二个细胞外段。而该家系中非患者和100名对照者基因组序列均无此改变。结论 位于GJA8 的c.565C>G突变是导致此先天性白内障家系可能的致病原因,缝隙连接蛋白第二个胞外结构域对晶状体的透明性起着重要作用。（眼科, 2013, 22: 86-89）     

Use of high-throughput targeted exome sequencing in genetic diagnosis of Chinese family with congenital cataract

AIM: To identify disease-causing mutation in a congenital cataract family using enrichment of targeted genes combined with next-generation sequencing. METHODS: A total of 371 known genes related to inherited eye diseases of the proband was selected and captured, followed by high-throughput sequencing. The sequencing data were analyzed by established bioinformatics pipeline. Validation was performed by Sanger sequencing. RESULTS: A recurrent heterozygous non-synonymous mutation c.130G>A (p.V44M) in the GJA3 gene was identified in the proband. The result was confirmed by Sanger sequencing. The mutation showed co-segregation with the disease phenotype in the family but was not detected in unaffected controls. CONCLUSION: Targeted exome sequencing is a rapid, high-throughput and cost-efficient method for screening known genes and could be applied to the routine gene diagnosis of congenital cataract.