不同载体对癌胚抗原DNA疫苗诱导小鼠产生抗—CEA水？…

目的 将癌胚抗原（ＣＥＡ）基因克隆入载体ｐｃＤＮＡ３及ＶＲ１０１２中，比较两套重组质粒在体内表达并诱导小鼠产生抗－ＣＥＡ的差别。方法 通过基因工程技术构建两套ＣＥＱ真核表达载体ｐｃＤＮＡ３－ＣＥＡ及ＶＲ１０１２－ＣＥＡ，将重组质粒肌注ＢＡＫＢ／ｃ小鼠，运用ＥＬＩＳＡ法检测不同时间肌肉组织表达ＣＥＡ水平及血清中抗－ＣＲＡ的产生。结果 ｐｃＤＮＡ３－ＣＥＡ在肌肉内呈低表达ＣＥＡ，最高时为０．１８ｍｇ／     

应用S基因转染的Sp2/0细胞作为靶细胞研究HBV基因疫苗的细胞免疫应答

构建编码ＨＢｓＡｇ 蛋白的重组真核表达质粒ｐＣＲ３－１Ｓ作为ＨＢＶ 基因疫苗, 免疫接种Ｂａ１ｂ／ｃ 小鼠。以重组质粒ｐＣＲ３－１Ｓ转染的Ｓｐ２／０ 细胞作为靶细胞, 采用５１Ｃｒ ４ｈ 释放法, 体外检测免疫小鼠的淋巴细胞杀伤功能。结果显示, 与空载体对照组相比较, ＨＢＶ基因疫苗可诱导Ｂａｌｂ／ｃ 小鼠产生ＨＢＶ 特异性细胞毒性Ｔ 细胞应答（ Ｐ＞ ０－０５） , 提示以Ｓｐ２／０ 基因转染的细胞作为靶细胞, 检测免疫Ｂａｌｂ／ｃ 小鼠淋巴细胞的杀伤功能是可靠的。     

应用S基因转染的Sp2／0细胞作为靶细胞研究HBV基因疫？…

构建编码ＨＢｓＡｇ蛋白的重组真核表达质粒ｐＣＲ３．１－Ｓ作为ＨＢＶ基因疫苗,免疫接种Ｂａｌｂ／ｃ小鼠,以重组质粒ｐＣＲ３．１－Ｓ转染的Ｓｐ２／０细胞作为靶细胞,采用＾５１Ｃｒ ４ｈ释放法,体外检测免疫小鼠的淋巴细胞杀伤功能。结果显示,与空载体对照组相比较,ＨＢＶ基因疫苗可诱导Ｂａｌｂ／ｃ小鼠产生ＨＢＶ特生细胞毒性Ｔ细胞应答,提示以Ｓｐ２／０基因转染的细胞作为靶细胞,检测免疫Ｂａｌｂ／ｃ小鼠淋巴细胞     

含有丙型肝炎病毒核心基因表达质粒的构建及其基因免疫

目的：研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫诱生特异性免疫应答的可行性。方法：将ＨＣＶ Ｃ基因片段插入真核表达载体ｐｃＤＮＡ３质粒ＣＭＶ启动子的下游,构建真核表达载体ｐｃＤＮＡＨＣＶ－Ｃ,分别转染小鼠骨髓瘤细胞ＳＰ２／０和人肝癌细胞７７２１进行瞬时表达,用免疫荧光法和Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测表达产物,将重组质粒注射,ＢＡＬＢ／ｃ（Ｈ－２＾ｄ）小鼠股四头肌,ＥＬＩＳＡ法检测血清中抗体产生水     

免疫刺激DNA及IL-2表达质粒对乙肝病毒DNA疫苗诱导抗体的影响

分别将含免疫刺激ＤＮＡ序列（ＩＳＳ）的质粒ｐＵＣ１９、ｐｃＤＮＡ３及编码ＩＬ ２的真核表达质粒ｐｃＤＮＡ３ ＩＬ ２,与乙肝病毒ＤＮＡ疫苗共同注射于小鼠胫前肌内。定量检测免疫小鼠体内ＨＢｓＡｇ的表达及血清中抗ＨＢｓ的水平。结果显示：ｐＵＣ１９、ｐｃＤＮＡ３及ｐｃＤＮＡ３ ＩＬ ２对乙肝病毒ＤＮＡ疫苗在肌肉内表达ＨＢｓＡｇ无影响或有抑制,但它们均能显著促进疫苗诱导抗体的产生（Ｐ＜００１）,抗体水平的增加与ＩＳＳ的数量呈剂量反应关系。     

基因免疫诱导9.1C3分子内影像类抗独特型抗体的产生

本文分别采用真核表达载体ｐＥＦ－ＢＯＳ及ｐＣＭＶ４构建含起始码和终止码的抗９．１Ｃ３分子单克隆抗体重链可变区基因重组表达质粒９．１Ｃ３ＶＨｐＥＦ－ＢＯＳ及９．１Ｃ３ＶＨｐＣＭＶ４，并将其分别免疫ＢＡＬＢ／ｃ小鼠。取免疫小鼠血清对Ｋ５６２细胞进行间接免疫染色和流式细胞仪分析表明，基因免疫小鼠体内可诱导９．１Ｃ３分子内影像类抗独特型抗体的产生。     

小鼠口眼HCV基因工程菌产生的细胞和体液免疫

近年来研究表明ＤＮＡ直接接种动物体内可诱导机体产生免疫反应，很可能成为预防感染的新型疫苗，即基因疫苗。含ＨＣＶ基因重组质粒ＤＮＡ的工程菌是否也有可能在预防ＨＣＶ感染中发挥一定的作用？本文初步观察了经口眼途径接种含ＨＣＶ核心基因重组质粒（ＨＣＶ，ｃｏｒｅ／ｐＭＡＬ）的大肠杆菌后，ＢＡＬＢ／ｃ小鼠体内的细胞和休液免疫反应。研究分为①实验组，分别口服１～３次（Ｈ１－Ｈ３）含ＨＣＶ．ｃｏｒｅ／ｐＭＡＬ的大肠杆菌；②口眼含ＰＭＡＬ质粒的大肠杆菌的对照组（Ｐ）；③不给细菌的对照组（Ｃ）。实验组小鼠淋巴细胞在…     

恶性疟复合多价抗原基因在小鼠中免疫应答的初步研究

目的探索恶性疟复合多价ＤＮＡ疫苗的可行性。方法把带有ＡＴＧ的接头与人工合成的恶性疟原虫复合多价抗原基因ＡＢ相连后，构建分别带有ＳＶ４０或ＲＳＶ启动子的真核表达载体ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ，重组表达质粒经肌肉注射免疫ＢＡＬＢ／ｃ小鼠后，检测其诱发特异性体液和细胞免疫应答水平及毒副作用。结果ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ免疫ＢＡＬＢ／ｃ小鼠后均诱发了一定水平的细胞及体液免疫应答，带ＲＳＶ启动子的ｐＲＥＰ９／ＡＢ免疫原性略强于带ＳＶ４０启动子的ｐＳＶ２／ＡＢ，ＤＮＡ免疫后未见明显的毒副作用。结论恶性疟复合多价ＤＮＡ疫苗可诱发特异的免疫应答，为疟疾ＤＮＡ疫苗的研究提供了一定的理论及实验依据     

基因免疫诱导9.1C3分子因影像类抗独特型抗体的产生

本文分别采用真核表达载体ｐＥＦ－ＢＯＳ及ｐＣＭＶ４构建含起始码和终止码的抗９．１Ｃ３分子单隆抗全重链可变区基因重组表达质粒９．１Ｃ３ＶＨｐＥＦ－ＢＯＶ及９．１Ｃ３ＶＨｐＣＭＶ４，并将其分别免疫ＢＡＬＢ／ｃ小鼠。取免疫小鼠血清对Ｋ５６２细胞进行间接免疫染色和流式细胞仪表明，其因免疫小鼠体内可诱导９．１Ｃ３分子内影像８类抗独特型抗体的产生。     

乙型肝炎病毒基因疫苗诱导小鼠产生特异免疫应答

构建编码乙型肝炎病毒（ＨＢＶ）表面蛋白Ｓ的重组质粒ｐＣＲ３．１－Ｓ。将之直接肌肉注射Ｂａｌｂ／ｃ小鼠,观察小鼠ＨＢＶ特异的免疫应答。以ＥＬＩＳＡ法检测小鼠血清,＾３Ｈ－ＴｄＲ掺入法测定淋巴细胞增殖,＾５１Ｃｒ４ｈ释放法检测淋巴细胞杀伤功能。结果表明,与空载体对照组相比较,基因疫苗诱发小鼠产生良了的抗ＨＢｓ反应及ＨＢＶ特异的细胞免疫应答（Ｐ〈０．０５）,提示基因疫苗ｐＣＲ３．１－Ｓ有可能成为控制ＨＢ     

Binding Reactivity of Monoclonal Anti-Carcinoembryonic Antigen (CEA) Antibodies with Cell Membrane-Bound CEA and with Free CEA in Solution

Binding reactivities of 62 anti-CEA MAbs from 10 different research groups with cell membrane-bound CEA and with free CEA in solution were compared by inhibition of MAb binding to CEA-expressing tumor cells by free CEA. Bindings of 30 MAbs to the cell membrane-bound CEA (280 ng CEA/2 × 10⁵ cells) were inhibited by approximately equal amounts of free CEA, indicating that binding affinities of about half the MAbs for cell membrane-bound CEA are similar to those for free CEA, respectively. Bindings of 15 MAbs to the cell membrane-bound CEA were easily inhibited by free CEA of less than half the amount of the cell membrane-bound CEA, while inhibition of bindings of the remaining 17 MAbs required twice more free CEA than the amount of cell membrane-bound CEA, showing that about one-fourth of the MAbs have higher affinities for free CEA and the remaining about one-fourth of the MAbs possess higher affinities for cell membrane-bound CEA. These results help form the basis for selecting the anti-CEA MAbs for use in clinical applications, such as serum CEA assay, tumor imaging and immunotherapy.     

Kinetic and affinity constants of epitope specific anti-carcinoembryonic antigen (CEA) monoclonal antibodies for CEA and engineered CEA domain constructs

Background: Carcinoembryonic antigen (CEA) is a human tumor antigen with the domain structure N-A1-B1-A2-B2-A3-B3, in which each domain is predicted to have an Ig-like fold and is known to bind epitope specific anti-CEA antibodies. Objective: To determine the affinity constants of several domain specific anti-CEA antibodies using purified recombinant or synthetic domains. Results and Conclusion: We have determined the kinetic and affinity constants of several anti-CEA antibodies for CEA, CEA domains (A3-B3) expressed in HeLa cells, and a synthetic peptide corresponding to the A3 domain using a BIAcore biosensor. There was no difference in affinity for CEA among a murine (mT84.66), a mouse/human chimeric form (cT84.66) or a disulfide deleted version (ΔSScT84.66) of this antibody. There was less than a five-fold drop in affinity of murine T84.66 for the A3-B3 domain expressed in HeLa cells compared to CEA. The synthetic A3 domain had an affinity constant for mT84.66 which was ten-fold less than for CEA. The affinity constants for CEA with several other anti-CEA monoclonal antibodies, including three antibodies which have almost identical CDR sequences (CEA.281, CEA.11 and CEM231) were also determined. CEM231 which had a two-fold higher affinity constant for CEA than either CEA.281 or CEA.11 had a two-fold faster on-rate which accounts for its higher affinity constant. This difference may be due to one or more of the amino acid differences present in H1 (N vs. S or D) and H3 (A vs. V).     

CEA Carcinoma Monitoring by Two RIA

A comparison was made of the accuracy of two CEA assays by RIA in diagnosis of different cancers. The basis of the study was a consecutive series of 76 patients thought to have bladder, colon, stomach or pancreas pathology, or a space occupying lesion; cancer was the final diagnosis in 46. The RIA assay method of Hoffman-La Roche and the CIS (Sorin) direct double antibody RIA assay were used for CEA detection. Results were correlated with the clinical and follow up evaluation. Samples were taken before operation, immediately after, and 1,2, 3 and 6 months after. Ninety eight per cent of the patients with a negative diagnosis for cancer had no detectable CEA, or levels below 2.5 ng/ml with Roche assay, and below 8 ng/ml with CIS assay. Abnormal CEA levels were found in the different groups as follows: with the Roche assay bladder—22%, colon—45%, stomach—23%, and pancreas—40%, with the CIS assay bladder—66%, colon—61%, stomach—38% and pancreas—60%. We conclude that neither test is ideal for clinical purposes although the CIS assay is probably more sensitive than the Roche assay. Both assays are useful in the monitoring and detection of recurrent tumors.     

CEA电化学发光免疫分析法的建立

目的建立人癌胚抗原（CEA）的电化学发光免疫分析法（ECLIA）。方法生物素标记的CEA McAb、钌复合物标记配对的CEA McAb、链霉亲和素包被的磁性微粒组成CEA ECLIA试剂,用ECLIA仪检测CEA并做方法学评价。用本法与进口的同类ECLIA试剂对119例结直肠癌、150例肺癌、45例胃癌、32例胰腺癌和34例肝细胞癌患者的血清CEA检测结果进行分析。调查218名志愿者血清CEA正常参考值。结果本法检测CEA的批内CV为1.6%～7.1%,批间CV为2.3%～11.7%,灵敏度为0.2 ng/mL。与CA19-9和AFP无交叉反应。自建ECLIA检测CEA浓度范围为0.2～1000.0ng/mL,正常参考值低于5.6ng/mL。结论自建CEA ECLIA与进口试剂比较（r=0.9641,P〈0.05）,正常参考范围接近进口同类方法。具备产业化的潜能。     

Analysis of the specificity of CEA reactive monoclonal antibodies. Immunological support for the domain-model of CEA

A panel of 17 monoclonal antibodies (MAbs), which are reactive with purified carcinoembryonic antigen (CEA), was tested. The MAbs were categorized into 6 groups according to their reactivity with CEA 180, CEA 160, non-specific cross-reacting antigen (NCA) 97 and NCA 50. After chemical modification of CEA (reduction, carboxymethylation, deglycosylation, enzymatic cleavage) and binding studies, the MAbs were further divided into 8 subgroups, representing 8 different antigenic sites on CEA. All MAbs bind to deglycosylated CEA. Most of the MAbs are directed against conformational determinants, since only three of them recognize reduced and alkylated CEA. The same three MAbs are able to detect 29 kDa glycosylated fragments obtained by enzymatic cleavage of CEA. These three protease V8- and trypsin-resistant fragments, probably obtained by interdomain cleavage, show a close relationship in peptide patterns, supporting the repeating structural domain-model of CEA as deduced from the cDNA sequence of CEA.     

接种CEA重组痘苗病毒对小鼠CEA^＋肿瘤抑制作用的机制探讨

目的 探讨CEA重组痘苗病毒（rV-CEA)治疗CEA^ 肿瘤的机制。方法 以我室构建的rV_CEA,腹腔接种供体C57/BL小鼠,取其脾细胞及腹腔巨噬细胞（Mφ）,分别过继转移给荷CEA^ -HePa肝癌细胞的C57/BL小鼠,检测该公共体小鼠脾细胞、腹腔Mφ及相应受体的脾细胞体外杀瘤细胞的效应,结果 接种rV-CEA的供体小鼠的脾细胞及腹腔Mφ过继免疫给受体小鼠,具有明显抑制受体CEA阳性肿瘤生长的作用,体外实验表明,该供体脾细胞及接种了供体Mφ的受体脾细胞对同一靶细胞的杀伤活性明显增强,但供体Mφ体外的细胞毒活性无明显增加,结论 rX-CEA对CEA^ 肿瘤的抑制作用,可能主要通过CEA特异性免疫反应激活T细胞而实现。Mφ作为抗原提呈细胞可通过激活T细胞而杀伤肿瘤细胞,具体机制值得进一步研究。