| The schistosomiasis is a kind of parasitic diseases,which can cause social and economic hardship. Although Significant progress has been made through drug of praziquantel thereapy in the control of schistosomiasis , hepatic fibrosis of chronic schistosomiasis has no effective therapeutic method. According to the network theory of the immune system of Jerne, Olds treat acute Schistosoma infection with anti-idiotypic antibody (anti-id), gaining low death rate, and the area of schistosomal granuloma grow down.The hybridoma cell of monoclonal anti-idiotypic antibody NP30 of Schistosoma japonicum has already been established by the Guan xiaohong professor. NP30 can induce protection immunity of certain degrees of mouse and goat actively immunized with NP30. But the NP30 is an antibody estabished from mouse.As an heterogeneity protein, it may cause human anti-mouse antibody (HAMA), which restrict in human application.With the development of technique for Antibody Engineering,we can reform mouse- antibody to decrease the harm to human body. Diabody is one kind of micromolecule antibodies,which has no constant region ,so it has low heterogeneity . Here we construct, express and characterizate a mono-specific diabody derived from monoclonal anti-idiotypic antibody NP30 of Schistosoma japonicum,and construct mouse models natural infected by Schistosoma japonicum cercaria, observe the treatment of NP30 diabody for the schistosomiasis. Methods1. The total RNA was extracted from the NP30 hybridoma cell, cDNA synthesized was gained from total RNA by reverse transcription. The variable region gene were amplified from the cDNA synthesized. The mono-specific diabody gene was constructed by overlap PCR and using Gly4Ser as a linker to join the VH to the VL.2. The diabody gene was linked with prokaryotic expression vector pBAD/gIII,and the recombinant was transformed to E. coli TOP10F'. The target protein expression was induced by L-Arabinose. Then a purification procedure for the target protein was carried out. The antigen binding activity of expressed protein was detected with ELISA..3. BALB/c mice were infected by schistosoma japonica cercaria, diabody were injected to infected mice at the second day, 2 weeks and 4 weeks after infection, and at the same time, control groups were set up. The death situation of each group at the 10th week after infection were observed and the still living mice were killed to count adult worms in each mouse and eggs in liver. Paraffin sections and HE staining of liver tissue of each mouse were carried out to analyze the average diameters and areas of all granulomas surrounding single egg. And Masson staining was used for ananlyzing levels of liver fibrosis.Results1. The mono-specific diabody gene was confirmed by sequencing,it was exactly similar with the variable region gene of NP30.The length of VH fragment was 351 bp,and the length of VL fragment was 318 bp.2. There were less soluble target proteins in the supernantes and higher target proteins in the pellets as inclusion body when separating the expression proteins.The molecular weight of target protein was ahout 34 kD.The expression proteins was purificated by His-Trap Ni affinity column .The binding activity of purificated protein with the blood serum of patients of Schistosoma japonicum was similar with NP30 antibody. 3. At the 10th week after infection,the death rates of Diabody group ,NP30 group and PBS group were 50%,27.8%,66.7% sepereately. The worm reduction rates of Diabody group and NP30 group were 19.25% and 29.07% , the death rate and adult worms of NP30 group were significantly lower than that of PBS group(P<0.05).Egg reduction rates of Diabody group ,NP30 group were 20.53% and 49.38%,they were bouth significantly lower than that of PBS group(P<0.05) . Image analysis showed that the average diameter and area of egg granuloma,levels of liver fibrosis of Diabody group and NP30 group were significantly lower than that of PBS group(P<0.05).Conclusions1. we construction, expression a mono-specific diabody derived from monoclonal anti- idiotypic antibody NP30 of Schistosoma japonicum.The constructed mono-specific diabody has similar affinity with monoclonal anti-idiotypic antibody NP30 of Schistosoma japonicum..2. The diabody can reduce the average diameter , area of egg granuloma and levels of liver fibrosis,which showing the diabody can decrease the immunopathologic damage of Schistosoma japonicum on host.
|
PDF Full Text Request