重组人G—CSF免疫小鼠全套单链抗体噬菌体表面展示文库的构建

利用全套噬菌体抗体表面展示技术,绕过杂交瘤技术,从重组人G-CSF免疫的小鼠脾淋巴细胞中提取总RNA,反转录成cDNA后,用抗体可变区PCR混合引物进行全套抗体重、轻链可变区（VH和VL）基因的扩增。经重叠延伸反应,在体外随机装配成单链抗体（ScFv）。将其克隆至噬菌粒载体pCANTAB5E中,电转化含SupE的E.Coli菌株,以辅助噬菌体M13K07超感染噬菌粒文库,构建成全套ScFv表面展示文库。为利用亲和富集筛选技术,获得具有G-CSF结合活性的完整重组噬菌粒克隆奠定了基础。     

重组人G—CSF免疫小鼠全套单链抗体噬菌体表面展示文库？…

利用全套噬菌体抗体表面展示技术，绕过杂交瘤技术，从重组人Ｇ－ＣＳＦ免疫的小鼠脾淋巴细胞中提取总ＲＮＡ，反转录成ｃＤＮＡ后，用抗体中变区ＰＣＲ混合引物进行全套抗体重，轻链可变区（ＶＨ和ＶＬ）基因的扩增，经重叠延伸反应，在体外随机装配成单链抗体（ＳｅＦｖ）将其克隆至噬菌粒载体ｐＣＡＮＴＡＢ５Ｅ中，电转化含ＳｕｐＥ的Ｅ．ｃｏｌｉ菌株，以辅助噬菌体Ｍ１３Ｋ０７超感染噬菌粒文库，构建成全套ＳｃＦｖ表面展示文     

抗P选择素免疫小鼠单链抗体噬菌体展示文库的构建和筛选

目的 构建抗P－选择素的全套单链抗体噬菌体表面展示文库，从中筛选与P－选择素高亲合力的单链抗体，为其临床应用奠定基础。方法 从P－选择素免疫的小鼠脾淋巴细胞中提取总RNA，反转录成cDNA后，用抗体可变区混合引物扩增全套轻、重链可变区基因，经重叠延伸反应，装配成单链抗体（ScFv)基因，将其克隆到噬菌体载体pHEN1中，构建单链抗体噬菌体抗体库。对抗体库进行亲合筛选后，ELISA法鉴定抗活化血小板单链抗体。结果 经过5轮“吸附－洗脱－扩增”的富集过程，phage-ELISA得到一个ELISA活性较高的与P－选择素结合的克隆。序列测定符合抗体可变区结构特点。结论 成功构建了全套抗P－选择素单链抗体噬菌体展示文库，并筛选得到具有P－选择素结合能力的单链抗体基因。     

抗血管性血友病因子A1区单链抗体噬菌体展示文库的构建和筛选

为构建抗人vWF-A1的全套单链抗体噬菌体表面展示文库,从中筛选与vWF-Al高亲合力的单链抗体,为其临床应用奠定基础。从vWF-A1免疫小鼠的脾淋巴细胞中提取总RNA,纯化mRNA并反转录成cDNA后,用抗体可变区混合引物扩增全套轻、重链可变区基因,经重叠延伸反应,在体外装配成单链抗体(scFv),将其克隆至噬菌体载体pHENl中,构建单链抗体噬菌体抗体库。对抗体库进行亲合筛选后,ELISA法鉴定抗vWF-A1单链抗体。结果经过5轮“吸附-洗脱-扩增”的富集过程,phage-ELISA筛选到与vwF-A1有高亲和力的克隆。序列测定符合抗体可变区结构特点。成功构建了全套抗vWF-A1单链抗体噬菌体展示文库,并筛选出具有结合vWF-A1功能的单链抗体。     

抗体库技术

1 初期的抗体库技术 抗体库技术即用基因工程的方法将全套抗体重链和轻链的可变区克隆出来,重组到原核表达载体,在原核体系中筛选特异性抗体的基因。它的出现标志着抗体技术发展的质的飞跃—从细胞工程抗体发展到了基因工程抗体时代,即第三代抗体。英国Chilswell     

杂交瘤细胞中的抗体可变区异常mRNA及去除

８０ 年代初期兴起的基因工程抗体技术具有很好的临床应用前景。其关键是对功能性免疫球蛋白（Ｉｇ）基因的可靠克隆。但在从杂交瘤细胞中克隆抗体的重链或轻链可变区（ＶＨ 或ＶＬ） 基因时， 常常会得到一些非功能性的、异常的序列。它们源自杂交瘤细胞中的抗体可变区（Ｖ 区） 异常ｍＲＮＡ。这些无效转录的ｍＲＮＡ 的存在， 可影响我们对正确抗体序列的克隆。本文就杂交瘤细胞中的抗体可变区异常ｍＲＮＡ 及去除方法进行了综述。     

丙型肝炎病毒非结构蛋白4A人源单链可变区抗体的筛选与鉴定

目的 利用噬菌体表面展示技术,从半合成人源化单链可变区抗体库中筛选、鉴定丙型肝炎病毒（ＨＣＶ）非结构蛋白ＮＳ４Ａ的单链可变区抗体（ＳｃＦｖ）及其编码基因,为抗ＨＣＶ的细胞内免疫基因治疗研究开辟新途径。方法 采用噬菌体表达展示技术,以重组的ＨＣＶ非结构蛋白ＮＳ４Ａ为包被抗原,从噬菌单链可变区抗体库中经过３轮“吸附－洗脱－扩增”筛选过程,获得抗原结合活性较强的ＨＣＶ ＮＳ４Ａ人单链可变区抗体段阳性克隆     

人源性抗乳腺癌Her-2/neu噬菌体单链抗体库构建

目的构建乳腺癌患者抗Her-2/neu全人源性噬菌体单链抗体可变区（ScFv）文库,筛选抗Her-2/neu单链抗体。方法收集40例乳腺癌患者前哨淋巴结（SLN）,提取总RNA,反转录为cDNA作模板,在目前常用的引物基础上重新设计可变区的引物,用PCR扩增全套抗体可变区,构建T载体库。并改造pCANTAB-5E构建了pCANTAB-Linker。将T载体库酶切连接入pCANTAB-Linker构建出ScFv文库。最后构建噬菌体单链抗体库并进行初级筛选。结果成功改良ScFv文库的构建方法。初步得到12株对人乳腺癌组织有高亲和力的Her-2/neu单链抗体。结论改良了ScFv文库的构建方法,可以获得较大库容量的单链抗体库,并从中筛选到人源性抗Her-2/neu单链抗体。     

抗溶藻弧菌独特型单克隆抗体单链抗体ScFv的构建和表达

目的：克隆抗溶藻弧菌独特型单克隆抗体ScFv。方法：从分泌抗溶藻弧菌独特型单克隆抗体的杂交瘤细胞株（2F4）中提取总RNA，利用RT-PCR技术，克隆该抗体重链可变区基因（VH）和轻链可变区基因（VL），通过基因重组构建VH-VL的表达载体pTAT-ALI，转化大肠杆菌BL21后用IPTG诱导表达。结果：VH基因序列全长369碱基对，编码123个氨基酸，VL基因序列全长339碱基对，编码113个氨基酸，二者均符合小鼠免疫球蛋白可变区基因特征，含有4个框架区（FRs）、3个抗原互补决定区（CDRs）及抗体特征性的两个半胱氨酸残基。ELISA测定显示基因工程抗体ScFv与原代抗体一样，具有较高的抗原亲和力。结论：成功构建了抗溶藻弧菌独特型抗体ScFv基因并表达于细菌壁膜间隙和包涵体中，为溶藻弧菌独特型抗体单链抗体ScFv成为新一代基因工程疫苗奠定了初步基础。     

单链抗体在基因治疗方面的应用

单链抗体（ｓｉｎｇｌｅ－ｃｈａｉｎ ａｎｔｉｂｏｄｉｅｓ）即众所周知的单链可变区片段（Ｓｉｎｇｌｅ－ｃｈａｉｎ ｖａｒｉ－ａｂｌｅ ｆｒａｇｍ ｅｎｔｓ，ｏｒ ｓＦｖ）是将抗体重链可变区和轻链可变区以一个弹性连接肽连接成为一个单个的多肽，它们能较好地保持原代抗体的亲合性和特异性，是一种新的基因治疗的手段。本文对使用单链抗体治疗癌症以及控制传染病的一些新的研究进展作一综述，并对这一技术的进一步应用进行了讨论。     

Level of functioning of siblings and parents of probands of varying degrees of retardation

A further analysis of already published data supports the position that retardates of low ability level less frequently have retarded siblings, retarded parents, and parents low in occupational level than do retardates higher in ability level. The analysis supports the position that there are two types of retarded individuals, persons retarded as a result of gene or chromosomal anomalies, brain injury, etc., who more frequently occur in the lower-level retardate group, and persons whose retardation represents polygenic segregation, who more frequently occur in the higher-level group.     

Modes of Inheritance of Errors of Refraction

Eighteen families in which both parents had refractions within the range of +4·0 D to −4·0 D and axial lengths seen in emmetropia (22·3-26·0 mm) showed coefficients of correlation of the order 0·5 indicative of polygenic inheritance. Such coefficients were seen for axial length (0·407) and for the cornea (0·487), but not for the lens (which is known to be yoked to the axial length). No such coefficients were seen in 19 families in which one of the parents had axial length outside the emmetropic range (nine families with long axes and 10 with short axes).

The pattern of polygenic inheritance for emmetropia (completely correlated optical components) and errors of refraction up to 4·0 D (inadequately correlated components: correlation ametropia) follows that seen in stature and other measurable characters. In contrast the high refractive errors with their abnormal axial lengths (component ametropia) are—like the extremes in stature—pathological anomalies with monofactorial inheritance.

     

Aspects of the problem of direct introduction of Standards of International Organizations

Editorial note. This article is published as part of a discussion. Particular issues of the article are disputable. First of all, this concerns the so-called “folder” method of introduction of international standards for medical devices to domestic medical practice (i.e., by direct translation of the standards and their publication as standardizing documents). Nevertheless, at least one of the problems, the problem of coordination between domestic state standards for medical devices and international recommendations of ISO and IEC, is undoubtedly of topical importance. Advancement of new health service legislation which is to be approved by law-makers will definitely introduce corrections into the present situation. The Editorial Board of Meditsinskaya Tekhnika believes this article will lessen these problems and to be welcomed by readers.