Production And Characterization Of Recombinant Human Monoclonal Antibody Fab Fragment Specific For Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular protozoan that infects an astonishing variety of vertebrate hosts including humans. Classified in the phylum Apicomplexa, T. gondii causes an opportunistic disease, toxoplasmosis, in individuals with immune dysfunction and congenital infection in infants. Antibodies are known to mediate resistance to T. gondii infection. The aim of our study is to prepare the human antibody Fab fragment against T. gondii for passive immunotherapy.In the present study we constructed a combinatorial immunoglobulin gene library from the peripheral lymphocytes of a patient with toxoplasmic encephalitis, and prepared the recombinant SAG1 as the target antigen for screening the library. After about 6×105 clones were screened the library by colony blotting, enzyme-linked immunsorbent assay and immunoflurescent assay, we got two positive Fab fragments specific to rSAG1, called as Tox08 and Toxll. The genes of two clones were sequenced and analyzed by IgBLAST. The heavy chain genes of Tox08 and Toxll were identify, belong to VH3-23, with 98% to 98.26% homology. In the light-chain genes of Tox08 and Toxll, the closest germ line V segment was Vκ1-33 or Vκ1-27, with 87.46% to 93.43% homology. To enhance the affinity, the light-and heavy-chain genes of Tox08 and Toxll were respectively shuffled and screened again. Two new clones named Tox87L-11H and Tox1403L-11H were got from a reshuffling library which was constructed by the heavy chain of Toxll and the light chain library from the patient with toxoplasmosis. Sequence analysis of the light-chain genes revealed that the nearest V-segment germ lines of Tox87L-11H and Tox1403L-11H belonged to Vκ1-17, with 92.11% and 89.61% homology respectivily. The three purified procedures were used to compare the quality of purified Tox1403L-11H. Through goat anti-human Fab fragment column, the proportion of the light chain and the heavy chain is 1:1, and yield 0.75mg/L Fab fragments. Through cobalt ions chelated column, the proportion of the light chain and the heavy chain is 1:2, and yield 1.16mg/L Fab fragments. Through nickel ions chelated column, the proportion of the light chain and the heavy chain is 1:1, and the production is 1.08mg/L Fab fragment. Affinity of Tox1403L-11lH to rSAGl was measured by surface plasmon resonance, which is purified with three columns. The dissociation constant was 2.01×10-8M, and the association constant was 9.0×107M. The affinity of Tox1403L-11H purified with nickel ions chelated affinity chromatography is better than others;It is the first report of gene analysis, bacterial expression and characterizing of human anti-SAG1 antibody from a combinatorial human immunoglobulin gene library constructed with the peripheral lymphocytes of a patient with toxoplasmosis. The purification process of recombinant ant-SAG1 Fab fragment was established. We belive that the present study will set stage for industrialization and the developing of the passive immunotherapy for toxoplamosis by using human engineering antibody.