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李先富, 唐家琪, 王长军, 白薇, 潘秀珍. 抗汉滩病毒McAb可变区基因克隆及序列分析[J]. 中国公共卫生, 2004, 20(1): 10-12.
引用本文: 李先富, 唐家琪, 王长军, 白薇, 潘秀珍. 抗汉滩病毒McAb可变区基因克隆及序列分析[J]. 中国公共卫生, 2004, 20(1): 10-12.
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LI Xian-fu, TANG Jia-qi, WANG Chang-jun, . Cloning and sequencing of variable region gene of McAb H7 againsthantaan virus[J]. Chinese Journal of Public Health, 2004, 20(1): 10-12.
Citation: LI Xian-fu, TANG Jia-qi, WANG Chang-jun, . Cloning and sequencing of variable region gene of McAb H7 againsthantaan virus[J]. Chinese Journal of Public Health, 2004, 20(1): 10-12.
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抗汉滩病毒McAb可变区基因克隆及序列分析

Cloning and sequencing of variable region gene of McAb H7 againsthantaan virus

    摘要
  • 摘要:
      目的   克隆和分析抗汉滩病毒单克隆抗体(McAb)H7可变区基因.
      方法   根据鼠源抗体可变区(V)氨基酸序列, 设计合成一组抗体重、轻链可变区(VH/VK)简并引物.采用一步法提取McAbH7细胞总RNA, 通过RT-PCR扩增V基因, 并进行核苷酸序列测定和分析.
      结果   通过RT-PCR成功克隆了H7可变区重轻链基因, 序列分析表明均为开放阅读框, 符合小鼠抗体框架结构, 含有明确的4个骨架区和3个抗原决定簇互补区, 与抗体二硫键形成有关的两个特征性半胱氨酸亦高度保守.同源对比表明, H7-VK长357bp, 编码119个氨基酸, 属JK4重排; H7-VH长360bp, 编码120个氨基酸, 属JH4重排.经GenBank查询证实为新基因, 登记号为AY245601和AY245602.
      结论   McAbH7可变区基因的获得, 为有目的地开展HFRSV基因工程抗体的遗传改造及体外亲和力成熟等研究奠定了基础.

     

    Abstract:
      Objective   To clone and analyze the variable gene of monoclonal antibody H7 againsthantaan virus.
      Methods   According to amino acid consensus sequences of mouse immunoglobulins(Igs)as described by Kabat et al, degenerated primers were designed and synthesized.The nucleotide sequences were derived by using the codons preferentially used in murine Ig variable region(V)genes as listed in the Kabat database.Based on RT-PCR technology, succeeding in preparing mRNA、amplifying cDNA and in cloning Ig heavy(VH)and light(VK)chain variable domains from murine hybridoma H7, which secreted antibody specifically binding to HFRSV, and the variable genes were cloned into T7-sequencing vector respectively.
      Results   the subsequent sequence analyses were performed with a DNA-STAR sequence analysis program, and the nucleotide and predicted amino acid sequences indicated that H7-VK gene consisted of 357 bp and 119 amino acids (JK4), 360 bp and 120 amino acids of H7-VH gene(JH4)respectively.The sequences were submitted to the GenBank database and they were given the following accession numbers: AY245601 and AY245602.
      Conclusion   the results would be potentially useful in constructing engineering antibody(such as genetic evolvement and affinity maturation in vitro)as thera-peutic reagents for HFRS patients.

     

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