Treatment of parvovirus infectious disease by a caninized neutralizing antibody to the virus

通过犬化病毒中和抗体治疗细小病毒感染性疾病

• 批准号: 13660330
• 负责人: IWATA Akira
• 金额: $ 2.11万
• 依托单位: Nippon Institute for Biological Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660330/
• 关键词: dog; parvovirus; treatment; CDR grafting; canonized; neutralization; monoclonal antibody; Contact determination; パルボウィルス; モノクローナル抗体

(1) Monoclonal antibody (mAb) against a canine parvovirus (CPV) and its cDNA from the hybridoma cells.CPV was propagated in CRFK cells and purified through density gradient ultracentrifugations. Hybridomas were obtained by immunization of mice with the purified CPV and subsequent fusion of spleen cells with P3U1 myeloma cells. After screening by ELISA, IFA and virus neutralization, six clones (CP1a-6a) were established. RNA was isolated from CP3a, which produced immunoglobulin (Ig) of G1 subtype and showed the strongest binding activity, then the cDNA of Ig light (L) and heavy (H) chain were cloned by PCR, and their nucleotide sequences were determined.(2) Cloning of cDNA of canine IgSpleen cells were isolated a beagle dog, and RNA was extracted, cDNA of Ig was isolated using primer designed from the nucleotide sequences reported previously. Four clones of H chain were consisted of about 470 amino acid residues (aa) and the constant regions have a homology of 60-70% to human or mouse Ig gamma H chains. Two clones of L chain (one kappa (242 aa) and one lambda (231 aa)) were obtained, which have a homology of 58% and 85% in constant region with human Ig, respectively.(3) Construction of a caninized antibody by complementary determining region (CDR) grafting.CDR was determined in H and L chain of the CP3a mAb cDNA by the Contact definition. The CDR of canine H and L chains was assumed by the same definition. The caninized Ig gene was designed by replacing the canine CDR with CP3a's. The DNA fragments of H and L variable domains were synthesized by PCR, and conjugated to the DNA encoding corresponding constant region of Ig, respectively. Both DNA fragments were inserted into a transfer vector and the recombinant baculoviruses were obtained. The expression of caninized Ig was demonstrated by anti-canine Ig antisem in the culture supernatant of the Sf21 cells infected with the recombinant baculovirus.

(1)从杂交瘤细胞中获得犬细小病毒（CPV）单克隆抗体（mAb）及其cDNA。CPV在CRFK细胞中繁殖，并通过密度梯度超离心纯化。用纯化的CPV免疫小鼠，然后将脾细胞与P3U1骨髓瘤细胞融合，获得杂交瘤。经ELISA、IFA和病毒中和筛选，建立了6个克隆（CP1a-6a）。从CP3a中分离出结合活性最强的G1亚型免疫球蛋白（Ig）的RNA，用PCR方法克隆Ig轻(L)链和重(H)链cDNA，并测定其核苷酸序列。(2)犬igcdna的克隆从beagle犬中分离脾脏细胞，提取RNA，利用先前报道的核苷酸序列设计的引物分离igcdna。4个H链克隆包含约470个氨基酸残基（aa），恒定区与人或小鼠Ig γ H链同源性为60-70%。获得了2个L链克隆（1个kappa （242 aa）和1个lambda (231 aa)），在恒定区与人Ig的同源性分别为58%和85%。(3)互补决定区（CDR）接枝法构建犬化抗体。通过Contact definition测定CP3a mAb cDNA的H链和L链上的CDR。犬齿H链和L链的CDR采用相同的定义。用CP3a代替犬的CDR基因设计犬化Ig基因。用PCR方法合成H和L可变结构域的DNA片段，分别与编码Ig恒定区对应的DNA进行共轭。将这两个DNA片段插入转移载体中，获得重组杆状病毒。重组杆状病毒感染Sf21细胞的培养上清中，用抗犬Ig反sem证实了犬化Ig的表达。