程序死亡蛋白1基因的克隆及原核表达

目的 克隆人程序死亡蛋白(PD-1)基因并构建PD-1蛋白的原核表达质粒,在大肠埃希菌中进行表达.方法 用RT-PCR的方法从慢性乙肝患者外周血淋巴细胞总RNA中逆转录得到PD-1基因的cDNA,构建PD-1基因的原核表达质粒,在大肠埃希菌BL21(DE3)中进行表达并纯化.用SDS-PAGE、DNA测序、氨基酸测序等方法对表达蛋白进行鉴定.结果 克隆到PD-1基因编码区全长序列cDNA,经DNA测序与已报道的序列同源性达99.8%.构建得到PD-1的原核表达质粒,并在大肠埃希菌中表达,纯化获得纯度较高的PD-1蛋白.氨基酸测序证明表达蛋白的正确性.结论 成功克隆人PD-1基因,在大肠埃希菌中获得表达,得到纯化的PD-1蛋白,为进一步研究PD-1蛋白的功能及应用打下基础.     

人T-bet基因的表达、纯化及其免疫原性分析

目的：利用载体pQE30在大肠埃希菌（M15）原核表达人T-bet基因全长序列，并对表达产物进行纯化、免疫动物和制备多克隆抗体。方法：利用PCR技术从克隆载体pGEM-T／T-bet获得人T-bet的全长编码序列，并将其亚克隆至原核表达载体pQE30，形成重组表达质粒pT-bet；酶切鉴定挑选阳性重组质粒转化大肠埃希菌JM109，测序鉴定后转化M15，经IPTG370C诱导4h后，SDS-PAGE电泳、考马斯亮蓝染色判断以包涵体形式存在的带有6×His标签的融合蛋白（表达产物），用Ni^2＋ -IMAC层析柱对融合蛋白进行纯化；将纯化的蛋白免疫BALB／c小鼠，制备人T-bet的多克隆抗体。结果：成功表达和纯化了人T-bet，并制备了多克隆抗体，ELISA和Western blot结果显示了血清抗体的高效价（1：100000）和高度特异性。结论：成功构建了原核表达载体pT-bet，并在工程菌M15中获得大量表达，经纯化后获得高纯度的人T-bet蛋白，制备了效价和特异性良好的多克隆抗体，为进一步研究T-bet的生物学功能奠定了的基础。     

重组人BMP-2的基因克隆及原核表达

目的 构建重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2)的原核表达质粒并在大肠埃希菌中诱导表达.方法 采用RT-PCR法,从骨髓细胞总RNA中扩增获得人BMP-2成熟肽cDNA,将其克隆入表达载体pBV220,构建BMP-2的重组原核表达质粒.重组质粒经酶切和测序鉴定后,在大肠埃希菌中诱导表达目的蛋白,表达产物采用Western印迹和ELISA进行鉴定.结果 测序表明,重组基因序列与人BMP-2成熟肽基因完全一致.Western印迹和ELISA检测显示,表达产物的相对分子量(Mr)与预期结果相符,与相应抗体有结合活性.结论 获得了人BMP-2成熟肽的编码基因,并构建了含有该基因的重组质粒pBV220/BMP-2,在大肠埃希菌中可高效表达人BMP-2.     

粉尘螨第五组主要变应原（Der f5）的克隆表达、纯化及免疫原性鉴定

目的克隆表达粉尘螨第五组变应原（Dermatophagoides farinae,Derf5）基因,并鉴定纯化蛋白免疫原性。方法提取活粉尘螨总RNA,扩增Derf5片段,PCR产物与克隆载体pMD18-T连接,转化入大肠埃希菌JM109,经酶切及测序鉴定获得pMD18-Der f5阳性菌株,再提取质粒进行双酶切,与表达载体pET-30a（＋）连接,转化入大肠埃希菌BL21,异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹法（Western blotting）鉴定其表达效果,Ni-IDA亲和层析柱纯化蛋白,利用尘螨病人血清鉴定其免疫原性。结果构建了重组质粒pMD18-Derf5和pET30a-Der f5,SDS-PAGE结果表明Der f5基因在BL21中获得良好的可溶性表达,蛋白质分子量与理论值相符,纯化的蛋白与病人血清有良好的IgE结合活性。结论获得广州地区Der f5的原核表达载体,高效表达纯化重组蛋白,初步鉴定了该蛋白的免疫原性。     

人干燥综合征B抗原重组体的构建及融合表达

目的克隆人干燥综合征B抗原基因(human sjogren’s syndrome antigen B,SSB)并进行原核表达,为使用重组抗原用于自身抗体的临床检测奠定基础。方法根据GenBank中检索到的人SSB cDNA序列,在5′非编码区和3′非编码区设计特异性引物,提取人源HeLa细胞总RNA作为模板,逆转录RT-PCR扩增人干燥综合征B抗原cDNA。PCR产物纯化后连接至载体PET-30a,导入大肠埃希菌DH5α,构建重组质粒PET-30a-SSB。对重组质粒进行酶切鉴定,选择阳性克隆测序。重组质粒导入大肠埃希菌BL21,阳性克隆经鉴定后在IPTG诱导下表达。结果RT-PCR扩增产物为1245bp。重组质粒PET-30a-SSB经EcoR Ⅰ和HindⅢ双酶切证实含目的基因片段。序列分析提示与GenBank中检索到的一致。SDS-PAGE和Western印迹结果显示融合蛋白相对分子量为73ku,具有天然SSB抗原活性。结论成功克隆SSB基因并表达融合蛋白。     

人β防御素4基因的合成及克隆载体的构建

目的：合成人β防御素4（HBD4）基因并构建其克隆载体.方法：根据GenBank中HBD4结构基因的序列, 设计合成了6条长度为32 ～60 bp的寡聚核苷酸片段, 用重叠延伸PCR法扩增合成HBD4 cDNA全长.PCR产物经双酶切后, 克隆入载体pMD18-T中, 并导入细菌中进行扩增.结果：HBD4基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、 测序分析证实, 合成的目的基因与设计的HBD4 cDNA的序列相一致, 并成功地克隆入克隆载体pMD18-T中.结论：用重叠延伸PCR法能方便、 准确地获得目的基因片段.重组克隆载体pMD18-T-HBD4 的获得, 为构建HBD4基因的表达载体并表达奠定了基础.     

丙型肝炎病毒非结构区NS3基因在大肠埃希菌中的可诱导性表达

目的 构建丙型肝炎病毒（HCV）NS3基因的原核细胞表达载体。实现在大肠埃希菌中的可诱导性表达。方法 应用聚合酶链反应（PCR）技术，以美国HCV-H株全长cDNA质粒为模板，扩增获得NS3基因片段，克隆到原核表达载体pET-30C( )中，构建原核表达载体pET-NS3，转化BL21（DE3）宿主菌，以IPTG诱导，获得NS3蛋白的可诱导性表达，以HCVNS3的单链可变区抗体（ScFv)证实表达的NS3蛋白的特异性，结果 以HCVNS3基因序列特异性引物，PCR扩增获得1893bp的NS3DNA征段，插入pET-30C( )表达载体，转化BL21（DE3）受体菌，经培养，IPTG诱导，获得了重组HCVNS3蛋白的表达，以HCVNS3的ScFv证实了表达的重组蛋白HCVNS3的特异性。结论 以大肠埃希菌表达了HCVNS3的重组蛋白质。     

Ⅱ型志贺毒素单克隆抗体可变区基因克隆及单链抗体的表达和功能鉴定

目的 从分泌抗肠出血性大肠埃希菌Ⅱ型志贺毒素中和单克隆抗体杂交瘤细胞株S2C4中克隆抗体可变区基因,构建单链抗体(ScFv),进行原核表达,并对其功能进行鉴定.方法 从杂交瘤细胞株S2C4中提取总RNA,逆转录成cDNA.在cDNA3'-OH末端添加poly-G.PCR扩增包括5'非翻译区和信号肽序列在内的抗体重、轻链可变区基因VH和VL,PCR产物装入T-A载体测序.根据测序结果,设计引物分别扩增VH和VL编码区,再通过重叠PCR,在VH和VL编码区基因之间引入连接链,构建ScFv基因,并克隆到表达载体pComb3xSS中.重组载体导入E.coli Top10F'进行表达,重组蛋白经纯化后,分别用ELISA和动物保护性实验鉴定其生物学活性.结果 VH和VL编码区基因全长分别为396 bp和378 bp,ScFv基因能在大肠埃希菌中高效表达,表达产物的分子量为34 000,用NiSO4亲和层析柱成功纯化.功能性实验表明纯化的重组蛋白可以与Stx2毒素有效结合,能保护动物抵御毒素分子的攻击.结论 成功地克隆S2C4单抗可变区基因,并构建、表达其单链抗体ScFv,为下一步进行该抗体人源化奠定实验基础.     

原核增强子样序列的筛选及其应用研究

目的 从大肠埃希菌C600株染色体基因组中筛选原核增强子样序列,构建携带原核增强子样序列的表达载体,探讨其对干扰素基因表达的影响.方法 采用氯霉素乙酰转移酶基因（CAT）作为报告基因,从大肠埃希菌C600株染色体基因组中筛选具有原核增强子样活性的序列,构建携带增强子样序列的表达载体,表达干扰素基因和检测干扰素活性.结果 从大肠埃希菌C600株染色体基因组中筛选到一个原核增强子样序列3A,其正、反向增强活性分别能提高β-半乳糖苷酶活性7.11和2.93倍.证实它的增强活性体现在转录水平;用原核增强子样序列3A的功能区3P3构建的表达载体,其表达的IFN-α2b型干扰素比原表达载体活性高3.7倍.结论 从大肠埃希菌C600株染色体基因组中筛选到一个原核增强子样序列3A,携带有原核增强子样序列的表达载体可提高干扰素基因的表达水平.     

人FOXP3 cDNA的克隆及在原核细胞中的表达

目的:克隆人FOXP3cDNA,构建其原核表达载体,在大肠杆菌中表达。方法:从新生儿脐带血获得FOXP3mRNA,用巢式RT-PCR技术扩增FOXP3cDNA,产物纯化后T-A克隆连接至pMD18-T载体,构建其原核表达载体pRSET-A-FOXP3,转化大肠杆菌BL-21(DE3)pLysS,IPTG诱导,表达产物经SDS-PAGE及Western blot检测和鉴定。结果:获得FOXP3mRNA,并对其编码区cDNA序列进行扩增。PCR产物连接至原核表达载体pRSET,经DNA测序,证实与Gen-Bank中的人FOXP3编码序列一致。经SDS-PAGE及Western blot显示在相对分子质量(Mr)约为52000处出现融合表达条带,表达量约占菌体蛋白总量的20%。结论:成功地克隆人FOXP3cDNA,构建了其原核表达载体,并在大肠杆菌中得到有效表达。     

Cleavage of human IgM with human lysosomal elastase

Human lysosomal elastase, a serine proteinase stored in the azurophil granules of polymorphonuclear leucocytes, cleaves human monoclonal IgM producing two fragments and dialyzable peptides. An F(ab)₂μ-like fragment, called IgMe in this report, retains some reactivity with an anti-Fcμ-antiserum and is antigenically deficient with respect to both the subunit (IgMs) produced by reduction and alkylation of IgM and the similar fragment (IgMp) produced by papain digestion. The other fragment is very similar to Fabμ generated by papain digestion, as indicated by immunochemical identity and a similar molecular weight.     

Novel human astroviruses: Novel human diseases?

Astroviruses are small, non-enveloped, single-stranded positive RNA viruses that belong to the Astroviridae family. While classical human astroviruses (HAstV) are a well-recognized cause of acute non-bacterial diarrhea among young children worldwide, novel astroviruses, named HAstV-MLB and HAstV-VA/HMO, have been identified recently in humans by molecular assays. They are phylogenetically more related to animal astroviruses than to classical human astroviruses, thus suggesting cross-species transmission. Serological studies demonstrated a surprisingly high seroprevalence in certain populations and highlighted a high infection rate in the early years of life. Although their pathogenic role has not yet been clearly determined, novel astrovirus RNA sequences have been identified in different biological specimens of symptomatic patients, including the feces, plasma, cerebrospinal fluid, and brain biopsies. Thus, there is evidence that they could contribute not only to digestive tract infection, but also to unexpected clinical syndromes, notably encephalitis and meningitis. Severe infections affect mainly immunocompromised patients. These findings indicate that novel astroviruses should be considered in the differential diagnosis of immunocompromised patients with meningitis or encephalitis of unknown origin.     

The binding of human IgG subclasses to human monocytes

The direct binding of human IgG subclasses to human monocytes has been measured by autoradiography using radiolabeled myeloma proteins. Only IgGl and IgG3 were found to bind strongly to the monocyte surface. This binding could be inhibited both by fresh human serum and by soluble immune complexes.     

Immortalization of human keratinocytes with human papilloma virus DNA

Human keratinocytes, derived from the cervix or foreskin, can be immortalized with the HPV-16 or HPV-18 E6 and E7 genes. Two methods of introducing the viral oncogenes into keratinocytes i.e. calcium phosphate transfection and retroviral transduction, are described below, both of which have been optimized for human keratinocytes. While the calcium phosphate transfection method can be used in a normal tissue culture facility, transduction with a retroviral vector containing oncogenes, requires a containment facility and appropriate laboratory practice.     

Persistence of human parvovirus B19 in human tissues

Human parvovirus B19 infection causes various clinical symptoms, such as rash, arthropathy, anemias and fetal death, but it can also remain asymptomatic. The arthropathies and anemias can become chronic for several years, not infrequently resembling autoimmune syndromes. B19 replicates only in red blood cell precursors of bone marrow or fetal liver, resulting in high-titred short-lived viremia, but viral DNA is detectable also in cells of several other types. Recently B19 DNA has been found, by very sensitive amplification tests, in certain tissues not only of symptomatic but also of healthy individuals for several years or decades after B19 infection. The mere presence of B19 DNA in these tissues of a symptomatic patient (e.g. joints in chronic arthritis or skin in dermatomyositis) thereby does not prove that the present disease is caused by B19. The diagnosis has to be verified by other innovative means. How and why viral DNA persists in the tissues of healthy individuals is under investigation.     

Detection of human cytomegalovirus DNA in human tonsillar lymphocytes

The human cytomegalovirus (HCMV) was first isolated in cell cultures from the oropharynx, which is thought to be a site of primary infection. Although HCMV can be recovered from the oropharynx during reactivation phases, its exact site of latency is not known. In the present study we demonstrated evidence suggesting the presence of latent HCMV in this anatomic region--in the palatine tonsils. Samples from 30 tonsils obtained by tonsillectomy were screened for the presence of HCMV. Out of the 30 tonsil donors, 23 were seropositive for HCMV. Three methods were used in attempts to demonstrate HCMV's presence in the tonsils: (1) viral isolation attempts on various cell cultures, (2) immunohistochemical staining--immunoperoxidase method--designed to detect viral antigens, and (3) DNA dot hybridization with a HCMV-DNA probe designed to detect viral DNA. Neither infectious HCMV nor other viruses were isolated in cell cultures. No viral antigens were detected by immunoperoxidase staining in the tonsillar tissue. Four out of the 30 tonsils studied were found to contain viral DNA. In one case in which the tonsillar mononuclear (MN) fraction was separated from the polymorphonuclear (PMN) fraction, only the first fraction contained the viral DNA.     

Development of human monoclonal antibodies against human cytomegalovirus.

Human monoclonal antibodies (HMAbs) against human cytomegalovirus (HCMV) have been developed by fusion of human spleen cells and human lymphoblastoid cell lines (NP101 and NP197). The cell line NP101 had great advantages in its high fusion frequency and the stability of the resultant hybridomas. The specificity of HMAbs was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Two of the six HMAbs obtained, which were IgG3 subclass, neutralized viral infectivity in the absence of complement. The neutralizing activity of one of these two HMAbs was enhanced in the presence of human complement, whereas the other was not. Another IgG1 subclass HMAb neutralized viral infection only in the presence of complement. The remaining three HMAbs showed no neutralizing activity. Those HMAbs may provide an important approach to studying human immune responses to HCMV. HMAbs having neutralizing activity may prove to be useful for passive immunotherapy of HCMV diseases.     

Effect of recombinant human interferon γ against human cytomegalovirus

Summary Escherichia coli-derived human interferon- (rIFN-) inhibited the replication of human cytomegalovirus (HCMV) synergistically when combined with IFN-. The induction of HCMV DNA polymerase was inhibited in rIFN--treated cells. It is suggested that the induction of 2–5 A synthetase does not play an important role in the anti-HCMV actions of IFNs.With 2 Figures