人rBPI23在大肠杆菌DH5α中的表达及其多克隆抗体的制备

目的制备人rBPI23蛋白并免疫家兔获得特异性多克隆抗体。方法将本室制备的pBV220-synBPI600表达载体转化感受态E.coliDH5α,温控诱导后,获得以包涵体形式表达的目的重组蛋白;用SDS-PAGE鉴定分子质量,West-ernblot鉴定抗原性;免疫家兔获得抗rBPI23抗血清,经饱和硫酸铵沉淀获得多克隆抗体,间接法ELISA检测抗体效价,Westernblot分析抗体的特异性。结果重组蛋白主要以包涵体形式表达,SDS-PAGE显示其分子质量约23ku,与预期结果相符;重组蛋白能与市售兔抗人BPI抗体特异性结合;免疫家兔获得高效价(1∶320000)抗血清,上述抗体能与rBPI23及人BPI标准品特异性结合。结论成功制备了人rBPI23,免疫家兔获得高效价抗人rBPI23多克隆抗体,为制备单克隆抗体及后期建立BPI免疫学检测方法奠定基础。     

人白细胞介素18 cDNA的克隆和在大肠杆菌中的表达

白细胞介素１８（ＩＬ－１８）是１９９５年克隆的一种新型细胞因子，又称ＩＦＮ－γ诱导因子，主要由活化的巨噬细胞产生，其相对分子质量（Ｍｒ）为１８　３００［１、２］。ＩＬ－１８可诱导ＴＨ１细胞产生ＩＦＮ－γ和ＧＭ－ＣＳＦ等细胞因子，增强ＮＫ细胞和ＣＴＬ的活性［１、２］，促进ＩＬ－２介导的Ｔ细胞增殖；增强ＴＨ１等细胞产生ＴＨ１类细胞因子［３、４］；促进免疫细胞表达ＦａｓＬ，增强Ｆａｓ介导的细胞毒作用等多种生物学功能［５］，在抗病原微生物感染﹑抗肿瘤及抗超敏反应等方面具有潜在的应用前景。因此，我们采用Ｒ…     

人G—CSF新型高效表达克隆的构建与鉴定

利用ＲＴ－ＰＣＲ技术，自人的脾脏单核细胞ｍＲＮＡ扩增出人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）结构基因，ＤＮＡ序列分析表明与天然ｈＧ－ＣＳＦ一致，将其克隆于表达载体ｐＪＧＷ１中，在大肠杆菌中进行诱导表达研究。结果表明：ｈＧ－ＣＳＦ重组蛋白表达率在２５％以上，并具有与天然ｈＧ－ＣＳＦ一致的生物学活性。     

抗华支睾吸虫抗体Fab段基因的克隆及重组表达载体的构建

为了制备抗华支睾吸虫基因工程抗体,本从感染华支睾吸虫患的淋巴细胞中提取总RNA,逆转录成cDNA。用相应的引物进行PCR,扩增出约700bp的重链Fd段和轻链к、λ基因,经XhoⅠ和SpeⅠ,SacI和XbaI双酶切后,分别和质粒载体pComb3连接,再经电穿孔转化大肠杆菌XL1-blue菌株,将轻链和重链Fd基因先后克隆入pComb3中,成功地构建了抗华丽支睾吸虫Fas段抗体基因的表达载体,为进一步构建噬菌体抗体库奠定基础。     

人淋巴细胞抑制素基因克隆与表达的研究

目的：获人SPN基因，构建SPN的原核表达载体。方法：采用RT－PCR的方法从激活的人外周血淋巴细胞的总cDNA中得到1493bp的人SPN基因，NdeI,BamH I双酶切后定向克隆到原核表达载体pET-11-c中，全自动测序仪测序，转化宿主菌BL21后，IPTG诱导，SDS－PAGE电泳分析。结果：成功克隆到人SPN基因，并构建表达质粒，SDS－PAGE电泳证实目的蛋白表达。结论：为进一步研究SPN的免疫抑制机理和作用以及探讨SPN作为新型生理性免疫抑制剂的可能性打下了坚实的基础。     

小鼠MBL-A基因的克隆和序列分析

目的：克隆小鼠甘露聚糖结合凝集素-A(MBLA)基因的全长编码区cDNA。方法：利用RT-PCR方法，从Balb/c小鼠的肝细胞中，分离出MBL-A基因cDNA片段，克隆入pUC-T载体，测序并进行分析。结果：扩增得到的小鼠MBL-A基因cDNA全长720bp，编码240个氨基酸残基，包含了完整的富含半胱氨酸区、胶原区、颈区和糖识别域。分析表明，与Genbank中发表的序列具有99.9％的同源性。结论：获得小鼠MBL-A基因的克隆，为进一步研究MBL-A分子在体内的生物学功能奠定了一定基础。     

人黑色素瘤抗原MAGE—3基因的克隆与表达

目的 克隆人黑色素瘤特异抗原MAGE-3基因,以制备肿瘤DNA疫苗。方法 用RT-PCR方法制备MAGE-3基因,以哺乳细胞高效表达质粒pCI-neo为载体,构建重组DNA疫苗。重组体用载体上的通用引物为测序引物,鉴定克隆的正确性。再将鉴定过的重组质粒用脂质体法转化293细胞,用免疫印迹法鉴定转化细胞中MAGE-3基因的表达。结果 正确构建了MAGE-3/pCI-neo重组质粒,并且在转化细胞中检测出了MAGE-3的表达。结论 成功地构建了重组MAGE-3/pCI-neo肿瘤核酸疫苗,可以进行下一步的肿瘤动物模型的疫苗接种及疗效观察。     

发现2个FGFR1细胞外段同源的cDNA片段

目的：克隆表达人成纤维细胞生长因子受体。方法：采用ＲＴ－ＰＣＲ法,结果：在钓取人ｃＤＮＡ的过程中,意外地钓取到２个与ＦＧＦＲ１细胞外段同源的ｃＤＮＡ片段,经克隆和ＤＮＡ序列分析发现它们是ＦＧＦＲ１细胞外源的ｃＤＮＡ片段,。结论：虽然对这个２个ｃＤＮＡ片段的功能尚不明确,但这种抓住新现象并进行探究的意识对科研很重要。     

Anti-neutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability increasing protein (BPI): a new seromarker for inflammatory bowel disease and associated disorders

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to be associated with ANCA, such as ANCA-associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary sclerosing cholangitis. The immunoreactivity of recombinant BPI (rBPI) was established in order to develop an ELISA specific for rBPI. By means of this assay, BPI-ANCA were assessed in sera of 178 patients with IBD or the associated disorder primary sclerosing cholangitis, 112 patients with ANCA-associated vasculitides, and in sera of 182 disease and 140 healthy controls. BPI-ANCA were found to be closely associated with IBD and primary sclerosing cholangitis (34% and 44% of ANCA-positive sera, respectively). By contrast, BPI-ANCA positivity was low (<10%) in the double-negative sera of patients with ANCA-associated vasculitides and in disease and healthy controls. BPI-ANCA appear to constitute an important marker for IBD and primary sclerosing cholangitis, but not for the ANCA-associated vasculitides.     

BPI N端优势抗原表位的模拟合成及其抗血清的制备

目的通过生物信息学模拟合成杀菌/渗透增强蛋白氨基端(BPIN端)优势抗原表位肽,免疫动物获得相应抗血清。方法利用生物信息学分析BPIN端(1-199)氨基酸序列的抗原性、亲水性、可塑性、表面可及性和二级结构等理化特性,据此设计合成TA/IK两条多肽,将其与钥孔戚血蓝素(KLH)偶联后,免疫家兔获得相应抗血清;采用间接ELISA法鉴定多肽的抗原性、测定血清抗体效价,Westernblot鉴定抗血清特异性。结果人工合成BPIN端TA/IK两个B细胞表位肽;ELISA检测证实TA/IK抗原肽能与商品化兔抗人BPI55多克隆抗体结合;所获TA/IK抗血清效价分别为1∶51200和1∶25600;Westernblot证实TA/IK抗血清能与BPI55标准品特异性结合。结论模拟合成的TA/IK抗原肽确为BPIN端优势抗原表位,相应抗血清可用于BPIN端功能性片段的检测鉴定。     

BPIm23重组抗菌蛋白在巴斯德毕赤酵母中的分泌表达

目的：采用巴斯德毕赤酵母表达系统表达分泌型功能性rBPIm23抗菌蛋白。方法：利用构建的pUC18-synBPI414质粒和PBV220-BPIm120质粒，按分子克隆方法构建pPICZa-synBPIm600重组表达载体；用线性化pVICZa-synBPIm600质粒电转化Pichia pastoris GS115，筛选抗性转化子，进行PCR和Mut表型鉴定；用甲醇诱导目的蛋白rBPIm23的表达；用离子交换层析纯化rBPIm23，并进行SDS-PAGE分析、Western blot鉴定和用BCA法定量。结果：①成功构建了pPICZa-synBPIm600重组表达载体；②获得稳定整合目的基因的GS115菌株；③表达产物相对分子量约为23kD，可与抗BPI抗体特异性结合；④培养上清液中目的蛋白表达量约为2.9mg／L。结论：BPIm23重组抗菌蛋白在毕赤酵母GS115中得到分泌表达。     

The bactericidal/permeability-increasing protein (BPI) is membrane-associated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation

Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability-increasing protein (BPI). BPI binds to lipopolysaccharides (LPS), and exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy. BPI was found to colocate with myeloperoxidase (MPO), a marker for azurophil granules, and it also showed the same pattern of distribution as CD63, a transmembrane-anchored protein. This suggests that BPI is membrane-associated in the azurophil granules in neutrophils. Its presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow. Induction of selective release of azurophilic granules by the Na-ionophore monensin resulted in fusion of endosomes with azurophil granules, leading to the formation of large vacuoles containing MPO, CD63, and BPI. After phagocytosis of serum-treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments. The results show that BPI is present in azurophil granules, is probably primarily membrane-associated, and is relocated after activation, following the same route as MPO and CD63.     

The DNA-bending protein, HMG1, is required for correct cleavage of 23 bp recombination signal sequences by recombination activating gene proteins in vitro

DNA-bending proteins are known to facilitate the in vitro V(D)J joining of antigen receptor genes. Here we report that the high-mobility group protein, HMG1, is necessary for the correct nicking of the 23 bp recombination signal sequence (23-RSS) by the recombination [corrected] activating gene (RAG) proteins, RAG1 and RAG2. Without HMG1, the mouse Jkappa1 23-RSS was recognized as if it were the 12-RSS and nicked at a site 12 + 7 nucleotides away from the 9mer signal, even though no 7mer-like sequence was evident at the cryptic nicking site. When increased amounts of HMG1 were added, the 23-RSS substrate was nicked correctly at a site 23 + 7 nucleotides from the 9mer, and nicking at the cryptic site disappeared. Unlike the 23-RSS, the 12-RSS did not require HMG1 for correct nicking, although HMG1 was found to increase the interaction between RSS and RAG proteins. Modification-interference assays demonstrated that HMG1 caused changes in the interaction between the 23-RSS and RAG proteins specifically at the 7mer and the cryptic nicking site.     

TnI(84-330bp)稳定区基因的克隆及表达

目的克隆与表达具有稳定免疫反应性的肌钙蛋白TnI（28—110aa）片段。方法（1）PCR从心脏cDNA中扩增TnI（84-330bp）与PGEX-4T-3／TnI（84-330bp）表达载体的构建；（2）IPTG诱导BL21-PGEX-4T-3／TnI（84-330bp）表达，根据聚丙烯酰胺凝胶电泳（SDS—PAGE）判定pGEx-4T-3-Tnl84—330bp融合蛋白的表达情况，并经蛋白质印迹试验（Western Blotting）对表达产物进行验证。谷胱甘肽Sepharose-4B亲合层析柱纯化目的蛋白。结果PCR扩增出TnI（84-30bp）基因，将该基因正确地克隆到表达性载体质粒pGEX-4T-3中；可溶性表达的目的蛋白多于包涵体；肌钙蛋白Ⅰ单抗能识别融合蛋白，而与GST蛋白不发生交叉反应。结论成功构建了能在BL21中高效表达的PcEX-4T-3／TnI（84—330bp）质粒，表达的目的蛋白有特异免疫反应性。     

Studies on the structure and function of the apolipoprotein(a) gene

Lp(a) is an LDL-like lipoprotein that is a major inherited risk factor for atherosclerosis. It is distinguished from Lp(a) by the addition of apolipoprotein(a). The gene structure of apolipoprotein(a) is homologous to plasminogen, and competition with plasminogen activity may account for some of the pathophysiology associated with Lp(a). Six highly related genes have now been identified, and at least four are found in close proximity in overlapping genomic clones. Studies have begun on the regulation of apolipoprotein (a) gene expression, and the human apolipoprotein(a) gene has been inserted into transgenic mice, where it leads to the development of arterial lesions.     

Cloning and sequencing of the 3-phosphoglycerate kinase (PGK) gene from Penicillium citrinum and its application to heterologous gene expression

The gene coding for 3-phosphoglycerate kinase (PGK) in ML-236B (compactin)-producing Penicillium citrinum was isolated from the recombinant phage lambda library using the corresponding Aspergillus nidulans pgk gene as a probe. The P. citrinum pgk gene has an open reading frame of 1,254 bp, encoding a protein of 417 amino acids with a predicted molecular weight of 44,079 daltons. The position of the two introns, 59 and 60 bp respectively, was deduced from an homology comparison with the sequence of the A. nidulans pgk gene. The PGK protein of P. citrinum shows extensive high homology to the PGKs of four other fungi: P. chrysogenum (93%), A. nidulans (84%), Trichoderma reesei (78%) and Saccharomyces cerevisiae (68%). Almost total conservation is found in P. citrinum of residues thought to be important for the structure and function of the yeast enzyme. The strong codon preference found has greater similarity to that in other filamentous fungi than in yeast. A DNA fragment encompassing the pgk gene was shown to hybridize a 1.35-kb poly(A)⁺RNA, sufficient to encode the PGK polypeptide. A fused gene, pgk-hpt, containing the putative pgk promoter and the open reading frame of the Escherichia coli hygromycin B phospho-transferase (hpt) gene was constructed, and was successfully used to transform P. citrinum to a hygromycin B (HmB)-resistant phenotype.     

Blepharophimosis sequence (BPES) and microcephaly in a girl with del(3) (q22.2q23): A putative gene responsible for microcephaly close to the BPES gene?

We report on a girl with the blepharophimosis sequence (BPES), microcephaly of postnatal onset, mild developmental retardation, and a deletion: 46,XX,del(3) (q22.2q23) de novo. A gene for BPES is suspected to be located at 3q23. Almost all cases with interstitial deletions containing 3q23 have not only BPES but also microcephaly and developmental retardation, while those without deletions, including those with apparently balanced translocations, only have BPES. Thus, a putative gene responsible for microcephaly may exist close to BPES gene. BPES, microcephaly, developmental retardation, and primary amenorrhea might constitute a contiguous gene syndrome. © 1993 Wiley-Liss, Inc.     

AngiostatinK（1—3）基因真核表达载体的构建,鉴定和表达

构建携带人ａｎｇｉｏｓｔａｔｉｎＫ（１－３）ｃＤＮＡ的真核表达载体,并将其在体外培养的脑胶质瘤细胞中表达。方法将带有分泌信号的ａｎｇｉｏｓｔａｔｉｎＫ（１－３）ｃＤＮＡ克隆入真核表达载体ｐｃＤＮＡ３,构建ＣＭＶ启动子控制的载体ｐｃＤ－ＮＡ－ＳＡＫ（１０３）,采用酶切鉴定结果。