抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达

目的 尝试将１株来源于人源性噬菌体抗体库的抗ＨＢｓＡｇ人Ｆａｂ,转换成完整的抗ＨＢｓＡｇ人ＩｇＧ。方法 采用重叠ＰＣＲ方法,在抗ＨＢｓＡｇ人Ｆａｂ和Ｖκ的ＶＨ基因的５’端接上人工合成的人κ链的前导序列,构建表达完整ＩｇＧ１的真核表达载体,转染ＧＨＯ（ＤＨＦＲ＾－）细胞,用ＥＬＩＳＡ、ＲＴ－ＰＣＲ和免疫印迹检测抗ＨＢｓＡｇ人ＩｇＧ的表达。通过ＤＨＦＲ／ＭＴＸ体系及金属离子诱导提高Ｉｇ表达。结果：获得     

人源性抗HBsAg单克隆抗体Fab片段的表达与纯化

对已筛选到的人源性抗ＨＢｓＡｇ特异性噬菌体抗体基因文库进行表达研究，将特异性噬粒转化大肠杆菌ＸＬ１－Ｂｌｕｅ，反复冻溶法制备可溶性Ｆａｂ片段。免疫印迹实验表明大肠杆菌质周腔内有可溶性Ｆａｂ片段的表达。制备羊抗人ＩｇＧ Ｆａｂ抗血清，建立抗人ＩｇＧ Ｆａｂ抗体－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析柱，纯化Ｆａｂ片段。ＳＤＳ－ＰＡＧＥ显示纯化的Ｆａｂ达免疫纯，点印迹法表达纯化后的Ｆａｂ片段具有中和ＨＢｓＡ     

抗人IgGFc和Fab单克隆抗体的鉴定及应用

本文将采用木瓜蛋白酶水解和ＳＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析等手段获得的人ＩｇＧＦｃ和Ｆａｂ，以抗人ＩｇＧＦｃ和抗人ＩｇＧＦａｂ单抗为参比品（Ｓｉｇｍａ）．鉴定了细胞库中抗人ＩｇＧ系列的部分细胞林，得到分泌特异性抗人ＩｇＧＦｅ和抗人ＩｇＧＦａｂ单抗的细胞各一株。在此基础上。应用抗人ＩｇＧＦｃ及抗人ＩｇＧＦａｂ单抗分别制备了Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱，纯化了酶解的人ＩｇＧＦｃ和Ｆａｂ。经ＥＬＩＳＡ法鉴定，相互间无交叉反应。同时用此方法还制备了人抗ＨＢｅＦａｂ，并将此Ｆａｂ标记过氧化物酶。配制ＨＢｅＥＬＩＳＡ诊断盒，证明其生物学活性未受影响，而且消除了类风湿因子引起的ＨＢｅＡｇ假阳性现象。     

BPI23-Fcγ1重组融合蛋白在E.coli中的表达

目的 构建ｐＢＶ－ＢＰＩ６００－Ｆｃγ１７００重组表达载体,转化Ｅ．ｃｏｌｉ ＤＨ５α诱导表达ＢＰＩ２３－Ｆｃγ１抗菌重组蛋白。方法 采用ＲＴ－ＰＣＲ技术,从ＨＬ－６０细胞和正常人白细胞ｍＲＮＡ中扩增编码ＰＢＩ２３和ＩｇＧ１Ｆｃ（Ｆｃγ１）的基因;通过连接反应构建重组克隆载体和重组表达载体;转化感受态Ｅ．ｃｏｌｉ ＤＨ５α细胞,通过温控诱导表达ＢＰＩ２３－Ｆｃγ１重组蛋白。结果 （１）ＲＴ－ＰＣＲ     

抗HBs人VH基因中异常序列对抗体活性影响的研究

目的 从噬菌体抗体库中克隆到１株ＶＨ第３骨架区中含有异常序列的抗ＨＢｓ人Ｆａｂ基因,探讨该ＶＨ基因中异常序列对抗体活性的影响。方法 采用重叠ＰＣＲ方法去除了这段异常序列,构建表达载体,转化大肠杆菌表达出抗ＨＢｓＡｇ噬菌体抗体和可溶性Ｆａｂ,测定了它们与抗原的结合活性。结果 与原抗体比较,改造后的抗体与抗原的结合活性明显下降,分子模建提示这段异常序列对ＶＨ ＣＤＲ１和ＣＤＲ２的部分氨基酸残基的位置有     

抗人IgGFc和Fab单克隆抗体的鉴定及应用

本文将采用木瓜蛋白酶水解和ＳＰＡ－Ｓｅｐｈａｒｏｅｓ４Ｂ柱亲和层析等手段获得的人ＩｇＧ Ｆｃ和Ｆａｂ，以抗人ＩｇＧ Ｆｃ和抗人ＩｇＧ Ｆａｂ单抗为参比品（Ｓｉｇｍａ），鉴定了细胞库中抗人ＩｇＧ系列的部分细胞株，得到分泌特异性抗人ＩｇＧ Ｆｃ和抗人ＩｇＧ Ｇａｂ单抗的细胞各一株。在此基础上，应用抗人ＩｇＧ Ｆｃ及抗人ＩｇＧ Ｆａｂ单抗分别制备了Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱，纯化了酶解的人ＩｇＧ     

抗菌肽MagaininⅡ单链抗体基因的构建及其表达

目的 将克隆的抗体轻重链可变区基因拼接成单链抗体基因并将其在大肠杆菌中表达。方法 通过ＲＴ－ＰＣＲ从两株抗ＭａｇａｉｎｉｎⅡ杂交瘤细胞株（２Ｄ１,３Ｆ８）中克隆出ＶＨ和ＶＬ基因,然后利用重组ＰＣＲ技术,将ＶＨ和ＶＬ基因通过柔性肽段（ＧＬＹ４Ｓｅｒ）３的Ｌｉｎｋｅｒ拼接成单链抗体基因（ＳｃＦｖ）,将ＳｃＦｖ克隆到表达载体ｐＣＡＮＴＡＢ５Ｅ上并将其分别在大肠杆菌Ｅ．ｃｏｋｉ ＨＢ２１５１及ＴＧ１中进行     

TNF-α单抗Fab段基因的克隆及其在大肠杆菌的表达

目的进行ＴＮＦ－α单抗Ｆａｂ段基因的克隆并在大肠杆菌中表达。方法用逆转录－聚合酶链反应技术（ＲＴ－ＰＣＲ）从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆重链Ｆｄ段和κ链基因；并用ＥＬＩＳＡ和免疫印迹分析证明表达的Ｆａｂ段特异结合ＴＮＦ－α。结果从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆出了重链Ｆｄ段和κ基因。经ＤＮＡ序列测定表明，ＶＨ、Ｄ、ＪＨ分别属于ＶＨ３Ｄ、ＤＳＰ２和ＪＨ４；Ｖκ和Ｊκ分别属于Ｖκ１和Ｊκ１。将该Ｆｄ和κ链ｃＤＮＡ克隆到表达载体ｐＣｏｍｂ３Ｈ中，在大肠杆菌中获得了表达。结论ＥＬＩＳＡ和免疫印迹分析表明，表达的Ｆａｂ段可特异地和ＴＮＦ－α结合。     

CLONING AND SEQUENCE CHARACTERIZATION OF THE RE-ARRANDED VH GENE FROM HYBRIDOMA CELLS SECRETING ANTI-HUMAN C1-INHIBITOR McAb

由分泌抗人Ｃ１－ＩＮＨＭｃＡｂ的杂交瘤Ｆ７细胞株提取总ＲＮＡ，合成与ＶＨ基因ＦＲ１和ＦＲ４互补的通用引物，以ＲＮＡ反转录合成的第一链ｃＤＮＡ为模板，ＰＣＲ法克隆出Ｆ７ＶＨ基因的ＤＮＡ片段。将分离获得的目的ＤＮＡ片段亚克隆入ｐＵＣ１８／１９测序载体，从两头进行双脱氧核苷酸随机终止法的ＤＮＡ序列测定。结果显示：ＶＨ基因是由３３３个碱基组成，编码１１１个氨基酸残基。通过国际联机检索进行ＥＭＢＬ和Ｋａｂａｔ基因库扫描发现，Ｆ７ＶＨＤＮＡ仅与Ｉｇ同源，符合小鼠Ｉｇ的ＶＨ基因特征；同源性为６０％～８５％，应归属于Ｉｇ的ＶＨ基因。根据Ｋａｂａｔ分类方法，Ｆ７ＶＨ基因推导的氨基酸顺序属于小鼠Ｉｇ的ＶＨ基因的Ⅱ（Ａ）亚组，是由ＶＨ－Ｄ－ＪＨ３重排产生；其框架区的９和６７位点为脯氨酸（Ｐｒｏ）和赖氨酸（Ｌｙｓ）（非芳香族氨氨酸），符合Ⅱ（Ａ）亚组框架残基结构模式；其ＣＤＲ３区含有７个氨氨酸残基，表明Ｃ１－ＩＮＨ抗原表面结构并不复杂；ＦＲ２和ＦＲ３的２２和８９位点为半胱氨酸残基（Ｃｙｓ），与ＶＨ片段二硫键形成有关。成功获得Ｆ７ＶＨ基因为进一步构建和表达单链Ｆｖ（ｓｃＦｖ）抗体打下良好的基础。     

随机化CDR3抗体库的构建及不经免疫制备抗体的初步探索

为探索利用噬菌体抗体库技术不经免疫制备抗体，在已有的抗乙肝病毒表面抗原（ＨＢｓ）人Ｆａｂ段基因的基础上，通过ＰＣＲ引入突变，对其ＶＨＣＤＲ３中１２个氨基酸残基（９５～１００Ｊ）进行了完全随机化，对９４和１００Ｋ位残基进行了有限随机化，构建了半合成噬菌体抗体库，库容为２×１０８，５８％含有随机化ＣＤＲ３序列，用无关抗原小鼠ＩｇＧ对该抗体库进行筛选，获得了三个可与小鼠ＩｇＧ结合而不与ＨＢｓ反应的克隆，经测定其ＣＤＲ３的ＤＮＡ序列，发现这三个克隆具有相同的ＣＤＲ３，证明单纯改变ＶＨＣＤＲ３可以得到新的特异性抗体，从而绕过免疫制备抗体。     

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.     

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.     

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.     

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.     

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.     

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.     

Uptake of human eosinophil peroxidase by human neutrophils.

A cytochemical analysis was carried out for study of the interaction between human eosinophil peroxidase (EPO) and human neutrophils. To this end, neutrophils with a genetic deficiency of myeloperoxidase (MPO) were used to avoid the otherwise inevitable interference of the high endogenous MPO activity of normal neutrophils. The data show that human neutrophils incubated with EPO (1 GU/ml) rapidly bind the enzyme all over the cell surface and internalize it in small vesicles. Part of bound EPO concentrates in a limited area on the cell surface and is then internalized by means of coarse tubular channels. Fusion of the small vesicles to each other or possibly with the tubular channels gives rise ultimately to EPO-containing multivesicular bodies, which, after 30 minutes of incubation, are the only peroxidase-positive structures in the cytoplasm. Under identical experimental conditions, no binding of human MPO to the neutrophils was detected. At concentrations 10 times as high as those used for EPO, a minority of neutrophils bound MPO, but the binding pattern remained diffuse on the plasma membrane and the internalization was negligible. It seems, therefore, that the EPO trapping system of human neutrophils exhibits specificity at least among leukocyte peroxidases. Furthermore, it operates at much lower concentrations of EPO than those reported for EPO uptake by mast cells and basophils. The uptake of EPO by neutrophils may serve to sequester a potentially toxic agent, thus limiting damage to the tissue in eosinophil-rich inflammatory lesions.     

Blastogenic response of human lymphocytes to human cytomegalovirus.

A method was developed for measuring the blastogenic response of human lymphocytes to human cytomegalovirus (CMV). Viral and control antigens were prepared by extracting disrupted infected and uninfected cell cultures with an alkaline buffer. Lymphocytes from ten donors with complement-fixing (CF) antibody exhibited a blastogenic response, whereas cells from ten seronegative donors did not. A relationship between the stimulation index (SI) and the results of neutralization (NT), indirect haemagglutination (IHA) or CF tests was not observed. The maximum blastogenic response occurred after 5 to 7 days of incubation and was usually greater when the cultures were supplemented with homologous plasma instead of sera. The presence of CMV antibody in the supplementary sera did not appear to affect the reactivity of the lymphocytes.