人细胞核自身抗原精子蛋白的重组表达及多克隆抗体制备

目的:获得纯化的人细胞核自身抗原精子蛋白(hNASP)及其多克隆抗体,为其功能研究做准备。方法:提取人睾丸组织总RNA,用自行设计的引物,PCR扩增hNASP的一段序列,PCR产物经TA克隆后,通过BamHⅠ和HindⅢ双酶切克隆到pET-28 a(+)中。在E.coliBL21中,用异丙基-β-硫代半乳糖苷(IPTG)诱导表达H is融合蛋白。样品超声处理后,经镍离子亲和树脂进行亲和层析纯化。用纯化的重组蛋白免疫家兔获取多克隆抗体。结果:对表达重组蛋白的质粒进行DNA测序以及表达的重组蛋白经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,证实获取了目的蛋白。ELISA证实免疫家兔后获得高效价的抗体。结论:用上述原核生物表达的方法可以得到纯化的hNASP蛋白,用纯化的蛋白免疫家兔也能获得高效价的抗体。     

改良型TAT-VP3融合蛋白的原核表达纯化及多克隆抗体的制备

目的 表达、纯化改良型TAT-VP3融合蛋白,并制备其多克隆抗体.方法 利用原核表达载体PGEX-6P-1/改良型TAT-VP3在大肠杆菌Rosetta(DE3)中表达,经GST标签纯化树脂纯化蛋白,并用PreScission Protease酶切除标签,以纯化的蛋白免疫新西兰大白兔,获得改良型TAT-VP3融合蛋白的多克隆抗体.用ELISA进行效价检测,Western blotting鉴定其特异性.结果 诱导表达并纯化了改良型TAT-VP3融合蛋白,纯度大于90%,蛋白浓度为1.2 mg/ml.制备了该蛋白的多克隆抗体,ELISA表明多克隆抗体效价为1∶32 000,Western blotting证明多克隆抗体的特异性良好.结论 成功纯化出改良型TAT-VP3融合蛋白,并制备出高效价、高特异性的多克隆抗体,为进一步研究该蛋白的体内外抗肿瘤活性及作用机制奠定了基础.     

人髓样细胞触发受体-1原核表达载体的构建和表达及多克隆抗体的制备

目的 构建人髓样细胞触发受体-1(TREM-1)原核表达载体使其表达并制备多克隆抗体。方法 采用特异性引物扩增人TREM-1胞外段cDNA,经酶切、连接、构建到原核表达载体pET28a(+),将构建的重组表达质粒转化入E.coli BL21( DE3)菌株,采用IPTG诱导表达、Ni-NTA 柱亲和层析纯化目的蛋白、SDS-PAGE分析蛋白纯度,将纯化的目的蛋白免疫家兔制备多克隆抗体,并对其进行纯化及鉴定。结果 序列测定证实构建的重组表达载体pET28a(+)-TREM-1含有人TREM-1编码序列,其序列分析与Genbank中公布序列对比一致,质粒在E.coli中诱导表达相对分子质量(Mr)为21.80 kDa的目的蛋白,SDS-PAGE分析表明纯化后的目的蛋白达到电泳纯,双向琼脂扩散法检测抗体效价为1:16,ELISA法检测抗体效价为1:25 600,Western blot分析显示抗体能特性结合人TREM-1重组蛋白。结论 成功构建高表达重组人TREM-1蛋白的原核表达载体及制备高效价兔抗人TREM-1多克隆抗体。     

DOC-1基因的原核表达载体构建及其重组蛋白的表达纯化

目的：克隆DOC-1基因、构建原核表达载体并纯化出其重组蛋白。方法：从人胎脑组织中提取总RNA，经逆转录一聚合酶链式反应（RT—PCR）扩增DOC-1的CDS序列，再通过基因重组技术将该基因片段依次克隆到pMO18-T和pGEX-4T-1载体中，构建融合表达载体pGEX-4T-1-DOC-1，经酶切、测序鉴定后，用该重组质粒转化E．coliBL21，用IPTG诱导表达，Glutathlone Sepharose 4B柱亲和层析纯化重组蛋白。结果：电泳证实RT-PCR扩增产物与预期目的基因DOC-1长度一致，测序结果与GenBank公布的DOC-1基因序列完全一致，IPTG诱导后经SDS—PAGE电泳分析表明，在相对分子质量38000左右出现新的蛋白表达条带，经亲和层析柱纯化后得到高纯度的GST-p12重组蛋白。结论：成功构建了pGEX-4T-1-DOC-1原核表达载体，表达并纯化出GST-p12重组蛋白，为进一步研究p12^DOC-1蛋白打下了实验基础。     

肝癌相关蛋白FAM172A2的克隆表达及多克隆抗体制备

目的 构建肝癌相关蛋白FAM172A2的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗FAM172A2蛋白多克隆抗体并进行鉴定.方法 应用逆转录-聚合酶链反应(RT-PCR)技术,以HepG2细胞总RNA为模板,扩增FAM172A2目的基因片段1113bp,构建原核表达载体,Western blot分析证实融合蛋白表达的特异性.诱导其大量表达后纯化、复性并制备多克隆抗体,Western blot及酶联免疫吸附试验(ELISA)检测特异性及其效价.结果 扩增获得FAM172A2基因片段,成功诱导FAM172A2融合蛋白表达并制备其多克隆抗体.ELISA检测证实其效价＞1∶160000,且特异性良好.结论 利用大肠埃希菌BL21( DE3)能够成功表达FAM172A2蛋白,获得高特异性、高效价兔抗FAM172A2蛋白的多克隆抗体.     

重组人中性粒细胞弹性蛋白酶的制备及鉴定

目的:利用基因工程技术制备纯化的重组人中性粒细胞弹性蛋白酶(HNE),为进一步制备HNE的相应抗体和建立精液HNE的检测方法奠定基础。方法:利用HNE的特异引物从人外周血粒细胞中获得HNEmRNA,并将其cDNA克隆入质粒pGEX-2T中以获得重组质粒pGEX-2T/HNE。重组质粒经PCR、双酶切和基因测序鉴定后转入感受态大肠埃希菌DH5α中,并用异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组融合蛋白GST/HNE。重组融合蛋白经凝血酶裂解后获得重组HNE,并经谷胱甘肽琼脂糖珠纯化后获得纯化的重组HNE。结果:成功制备重组表达质粒pGEX-2T/HNE,并转化入大肠埃希菌DH5α中。经IPTG在18℃过夜诱导后成功获得重组融合蛋白GST/HNE的表达。经凝血酶裂解和谷胱甘肽琼脂糖珠纯化后成功获得纯化的重组蛋白HNE。结论:纯化的重组HNE的获得为进一步制备HNE的相应抗体和建立精液HNE的检测方法奠定了基础。     

MGP基因的重组及在大肠杆菌中的表达

目的重组骨质疏松候选基因基质Gla蛋白（MGP）基因使其蛋白在大肠杆菌中高效表达。方法利用反转录聚合酶链反应（RT—PCR）从正常人肺组织总RNA中扩增出MGP基因cDNA序列，与克隆pGEM—Teasy载体相连，测序为完整的编码序列后与表达载体pTrcHisB构建重组体，转化入大肠杆菌Top10后用IPTG诱导，Western bloting证实蛋白表达。结果克隆至pGEM-Teasy载体及pTreHisB载体中的MGP基因cDNA序列与基因库完全一致。转入大肠杆菌后经IPTG诱导有蛋白的表达，Western bloting证实诱导后2、3、4h蛋白的表达量显著增加。结论成功重组的人MGP基因，重组体在大肠杆菌内能成功高效地表达。IPTG诱导后蛋白的表达为时间依赖性。     

Cyr61蛋白表达载体的构建及表达

目的：体外表达并纯化Cyr61蛋白片段。方法：提取乳腺癌组织总RNA，用一步法RT—PCR选择扩增编码Cyr61蛋白的cDNA片段，并将之克隆到融合蛋白表达载体pQE80L中，转染大肠杆菌Rosetta—gami^TM2。异丙基硫代半乳糖苷（IPTG）诱导表达融合蛋白，用亲和层析法纯化。纯化产物经聚丙烯酰胺凝胶电泳及蛋白质印迹分析鉴定。结果：克隆到编码Cyr61蛋白片段的cDNA片段，其大小为930bp。构建的表达质粒PQE80-Cyr61经限制性内切酶酶切和DNA测序证实为所需要的质粒。表达出相对分子质量分别为32kD的可溶性融合蛋白，经蛋白质印迹分析鉴定为Cyr61的融合蛋白。结论：本实验克隆了编码Cyr61蛋白片段的cDNA序列，并成功地获得了可溶性表达的融合目的蛋白，为进一步制备抗Cyr61蛋白的抗体和建立Cyr61蛋白定量检测方法创造了条件。     

FAM172A及其异构体重组蛋白对体外培养肝细胞增殖的影响

目的体外克隆人类基因FAM172A,构建其原核表达载体并诱导其重组蛋白的表达,制备兔抗FAM172A重组蛋白的多克隆抗体,观察其在不同细胞系的表达情况。观察FAM172A-1和FAM172A-3蛋白对L02细胞增殖的影响。方法利用反转录聚合酶链反应（RT-PCR）及PCR技术,构建原核表达质粒pET-32a（＋）-FAM172A-1和pET-32a（＋）-FAM172A-3。诱导该基因不同异构体重组蛋白的表达,并通过蛋白质免疫印迹（Western blot）技术进行鉴定。纯化后的重组蛋白FAM172A-1和FAM172A-3与肝细胞L02细胞共孵育,观察不同浓度的重组蛋白对细胞增殖的影响。利用纯化后的FAM172A（异构体3）重组蛋白免疫大耳白兔,获得抗FAM172A-3蛋白的多克隆抗体,利用酶联免疫吸附法（ELISA）以及Western blot技术对获得的多克隆抗体进行效价分析及特异性检测。结果成功扩增获得FAM172A（异构体1、3）的基因片段,测序结果与GenBank已公开的基因序列一致;成功表达FAM172A（异构体1、3）的重组蛋白,经过Western blot鉴定正确;纯化后的重组蛋白FAM172A （异构体1、3）与L02细胞共孵育结果发现,两者对L02细胞在一定浓度范围内均有促进细胞增殖的作用。所制备的兔抗人FAM172A-3重组蛋白的多克隆抗体,ELISA检测显示其效价可达1︰1280000, Western blot检测证实该多克隆抗体的特异性良好;Western blot分析显示,该蛋白在肝脏间质细胞和实质细胞均有一定程度的表达。结论 FAM172A蛋白在肝实质细胞及肝间质细胞均表达,且其重组蛋白可以促进L02细胞增殖,推测该基因可能与肝细胞损伤、肝纤维化以及肝细胞再生等发生机制有关。     

免疫调控相关基因C12orf28在免疫组织中的分布特征

目的：制备抗人C12orf28多克隆抗体,明确C12orf28编码蛋白在免疫组织中的分布特征。方法选取免疫原性高、特异性强的羧基端257个氨基酸作为目的片段,以人外周血单个核细胞（PBMC）cDNA为模板,采用PCR获得目的基因C12orf28C257;构建其原核表达载体pET32a（＋）-C12orf28C257,转化入大肠埃希菌BL21（DE3）,异丙基β-D-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白;将重组蛋白免疫大耳白大白兔制备兔源性多克隆抗体,采用ELISA和Western blot分析检测多克隆抗体效价和特异性,采用Western blot明确C12orf28的组织分布特征。结果 PCR扩增得到C12orf28C257目的基因片段,诱导表达重组蛋白并制备了其多克隆抗体。ELISA分析结果显示,多克隆抗体效价＞1︰1.28×106,Western blot分析鉴定得出多克隆抗体具有较高的特异性。结论未知功能基因C12orf28在胎儿肠、淋巴、胸腺和心肌等组织中均有不同程度的表达。     

人精子表面蛋白P34H重组表达载体的构建及诱导表达

目的 :获得纯化的人精子表面蛋白P34H用于基础和临床研究。　方法 :在克隆到P34H基因编码区全长的基础上 ,将P34H基因亚克隆至原核表达载体pQE 30中 ,构建重组表达载体pQE 30 /P34H。将pQE 30 /P34H转化至大肠埃希菌后 ,用异丙基 β D 硫代半乳糖苷 (IPTG)诱导表达 ,通过Ni NTA树脂进行亲和层析纯化上清中可溶性形式的重组蛋白P34H。对表达纯化重组蛋白的质粒进行DNA测序 ,并对重组蛋白P34H行十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)分析 ,鉴定其是否为所需目的蛋白。　结果 :经PCR和双酶切鉴定 ,重组表达载体pQE 30 /P34H为正确克隆。SDS PAGE和DNA测序证实 ,所获的重组蛋白确系所需目的蛋白。　结论 :用上述原核表达的方法可获得纯化的人精子表面蛋白P34H。     

一种新型兔源内毒素结合蛋白的分离纯化及体外生物活性的初步研究

目的　分离纯化一种新型的内毒素结合蛋白 ,并初步观察其生物学特性。　方法　建立兔烧伤合并内毒素血症模型 ,采其血清 ,经二步离子交换层析及凝胶过滤分离纯化 ,并经流式细胞仪分析、绵羊红细胞凝聚试验、氨基末端氨基酸残基测序等方法鉴定。将所获蛋白质作用于体外培养的人单核细胞 (U937) ,观测其分泌TNFα等细胞因子的状况。　结果　所获蛋白质的分子量为 48kDa( 48× 10 3) ,其N端 10个氨基酸序列为GSQGTFTSEE ,与美国国立图书馆蛋白质库中有关兔的氨基酸序列进行比较 ,未发现相同序列 ,暂命名为P48。它具有与内毒素结合蛋白相似的功能 ,能促进极低浓度的脂多糖 (lipopolysaccharide ,LPS)与外周血单核细胞 (PBMC)结合 ,并促进U937分泌TNFα。　结论　从兔血清中能够分离纯化出带有生物学活性的新型内毒素结合蛋白P48,它具有与脂多糖结合蛋白 (lipoplysaccharidebindingprotein ,LBP)相似的生物学活性 ,并可促进炎症反应的发生。     

Purification of testosterone-oestradiol-binding globulins from mammalian sera by anion-exchange high performance liquid chromatography

Testosterone-oestradiol-binding globulin (TeBG) has been isolated from serum or plasma of several species using procedures that yielded highly purified protein, but which required multiple and tedious chromatographic steps. In this report we describe a procedure for the isolation of TeBG which involves two chromatographic steps: androgen affinity chromatography followed by anion-exchange high performance liquid chromatography (anion-exchange HPLC). The purity of the final product was confirmed by silver staining following fractionation on sodium dodecyl sulphate-containing polyacrylamide gels. The size heterogeneity and specific binding activity of TeBGs purified from human, rabbit, or bull serum (or plasma) by this technique was indistinguishable from preparations obtained by conventional chromatography. The present technique shortened the entire purification procedure to about 5 working days and yielded milligram quantities of highly purified protein. Bases on our experience with serum or plasma from the human, rabbit, and bull, this approach should be suitable for isolation of TeBG from a wide range of species.     

Development of a heterologous radioimmunoassay for canine osteocalcin

The aim of this study was to develop a routine and reliable radioimmunoassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were purified to apparent homogeneity according to N-terminal sequence analysis. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated molecule. High titer (>1:5,000) anti-dog osteocalcin antisera were produced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat. A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantages of the facts that (1) anti-dog osteocalcin crossreacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tracer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and diluted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30–40 ng/ml rat osteocalcin-equivalent. The inter-and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteocalcin in serum. The results quantitated with this RIA correlated well (r-0.975, n=86) with those obtained with the homologous RIA. Application of the heterologous assay to dogs of different age revealed that young dogs (3 months old) had 15-fold higher serum osteocalcin level than adult (>2 years old) dogs. In summary, we have (1) purified dog osteocalcin; (2) produced an antiserum against it; and (3) developed a heterologous RIA that could accurately measure dog osteocalcin, and could be used routinely to measure dog osteocalcin.This work was presented in part at the annual meeting of the American Society of Bone and Mineral Research in Minneapolis, September 1992.     

The effects of acidic epididymal glycoprotein (AEG) and some other proteins on the motility of rat epididymal spermatozoa

Acidic epididymal glycoprotein (AEG) had a slight stimulatory effect on the motility of spermatozoa showing low initial motility which had been removed by micropuncture from the caput and corpus epididymidis of rats. However, similar effects were seen when bovine serum albumin (BSA) or purified gammaglobulin against AEG was used instead of AEG. Furthermore, BSA, normal rabbit serum and serum from a rabbit immunized against AEG reduced the motility of spermatozoa which showed high initial motility after removal from the caput, while AEG had no effect. These studies emphasize the importance of the effects of proteins on the motility of spermatozoa but do not provide any clear evidence for a specific effect of AEG.     

Heterogeneity in end-terminal sugars of rabbit and rat androgen binding protein (ABP)

The interaction between rabbit and rat androgen binding protein (ABP) and rabbit serum testosterone binding globulin (TeBG) with concanavalin A (Con A) was studied using affinity chromatography on Con A-Sepharose 4 B columns. When partly purified rat ABP, equilibrated with [³H]5α-dihydro-testosterone [³H]DHT was applied to Con A-Sepharose columns, approximately 50% of the ABP was retained by the column, whereas the remaining was eluted with the break-through protein fraction. A similar picture was found using partly purified rabbit ABP, or crude rabbit rete testis fluid. These studies indicate that both rat and rabbit ABP are glycoproteins, showing heterogeneity in their end-terminal sugars. When partly purified rabbit TeBG was examined by Con A-Sepharose affinity chromatography, the TeBG was completely retained by the column. The different elution patterns between rabbit ABP and rabbit TeBG indicate that these proteins, although showing identical physico-chemical and immunological properties (Weddington et al. 1975a, b). possess differences in their carbohydrate content.     

表达前强啡肽基因的Ad5型腺病毒载体的构建及鉴定

目的 构建且鉴定含前强啡肽基因(prodynorphin)的血清5型腺病毒载体(Ad5).方法 应用分子生物学方法 将前强啡肽基因序列克隆入腺病毒穿梭质粒pDC316-PDP,后者与骨架质粒共转染HEK293细胞,包装得到含前强啡肽转基因的腺病毒Ad5-PDP.用聚合酶链反应(PCR)方法 对转基因病毒进行鉴定,TCID50法测定病毒滴度.结果 PCR法证实转基因正确插入了Ad5型病毒基因组内,且没有野生型病毒污染,病毒滴度为1×1012v.p./mL.PCR鉴定Ad5-PDP重组成功.结论 获得的Ad5-PDP滴度高,感染性好,可以用于转基因治疗的实验研究.     

Antisera to a rabbit urinary tract antigen also react with human bladder and kidney tissue

The mucin layer covering the bladder transitional cell mucosa appears to function as a primary defense mechanism against bacterial infection. We have previously prepared a glycoprotein fraction (GP1) from the urinary bladder mucosa of NZW rabbits and raised murine antisera against it. These antisera react with bladder, ureter and kidney tissue from rabbits, rats, guinea pigs, and hamsters. We now show that a similar substance occurs in human kidneys and bladder. In order to remove antibodies reactive with the Tamm-Horsfall protein (THP), the antisera were initially absorbed with an immunoadsorbent composed of purified human THP covalently bound to Sepharose CL-4B gel. Using an enzyme linked immunosorbent assay (ELISA) it could be shown that the absorbed antisera did not react with THP but retained a high titer in binding to GP1. Immunohistochemical procedures involving avidin-biotin-immunoperoxidase staining demonstrated that the absorbed anti-GP1 reacted well with six human urinary bladder biopsy specimens and two kidney autopsy specimens while normal murine sera showed little or no binding. Although this reactivity was not as strong as that found with homologous tissue (rabbit) these studies suggest that GP1, an antigen common to several animal species, is also related to a human urinary tract component.