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

(1)从杂交瘤细胞中提取抗犬细小病毒(CPV)的单抗及其基因，在CRFK细胞中扩增，密度梯度超速离心法纯化。用纯化的CPV免疫小鼠，然后将脾细胞与P3U1骨髓瘤细胞融合，获得杂交瘤细胞。经EL ISA、IFA和病毒中和筛选，获得6个克隆(CP1a-6a)。从结合活性最强的G_1亚型免疫球蛋白(Ig)的Cp3A中提取核糖核酸，用聚合酶链式反应方法克隆了免疫球蛋白轻链(L)和重链(H)的基因，并测定了它们的核苷酸序列。(2)克隆犬免疫球蛋白细胞的基因，提取核糖核酸，根据已报道的核苷酸序列设计引物，获得免疫球蛋白基因。四个克隆的H链由约470个氨基酸残基组成，其恒定区与人或鼠的Ig-Gamma H链有60-70%的同源性。获得了两个L链克隆(一个kappa(242aa)和一个lambda(231aa))，它们在恒定区与人Ig的同源性分别为58%和85%。(3)通过互补决定区嫁接构建角化抗体。犬H链和L链的CDR按相同的定义假定。用Cp3A‘s取代犬CDR，设计犬化Ig基因，用聚合酶链式反应合成H和L可变区，分别与相应的Ig恒定区连接。将这两个DNA片段插入转移载体，获得重组杆状病毒。在重组杆状病毒感染的Sf21细胞培养上清中，用抗犬Ig抗体证实了犬化Ig的表达。

(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.