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

(1)本文报道了抗犬细小病毒（CPV）的单克隆抗体（mAb）及其cDNA。用纯化的CPV免疫小鼠，然后将脾细胞与P3 U1骨髓瘤细胞融合，获得杂交瘤。经过ELISA、IFA和病毒中和筛选，建立了6个克隆（CP 1a-6a）。从产生G1亚型免疫球蛋白（IG）且结合活性最强的CP 3a中提取RNA，通过PCR方法克隆了IG轻链（L）和重链（H）的cDNA，并测定了其核苷酸序列。(2)犬免疫球蛋白cDNA的克隆从Beagle犬脾细胞中分离RNA，根据已报道的核苷酸序列设计引物，从脾细胞中分离出IG的cDNA。4个克隆的H链由约470个氨基酸残基（aa）组成，其恒定区与人或小鼠IG γ H链的同源性为60-70%。获得了两个L链克隆（一个κ（242 aa）和一个λ（231 aa）），其恒定区与人IG的同源性分别为58%和85%。(3)用互补决定区（CDR）移植法构建了CP 3a单抗cDNA的互补决定区（CDR）。犬H和L链的CDR采用相同的定义。通过用CP 3a替换犬CDR来设计犬源化的IG基因。通过PCR合成H和L可变区的DNA片段，并分别与编码IG的相应恒定区的DNA缀合。将这两个DNA片段插入转移载体，获得重组杆状病毒。用重组杆状病毒感染Sf 21细胞，培养上清中抗犬IG抗体阳性，证明表达了犬源化IG。

(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.