| Rabies is known as an infectious disease.The chied criminal is caused by rabies virus,once human and animal are infected with the disease,the death rate almost reach 100%.Post exposure prophylaxis(PEP)is the best way to control the disease.Post exposure prophylaxis consists of three steps:cleaning the wound,vaccination and infiltration of the immunoglobulin.Because there are the very large side effects of the existing rabies immunoglobulin serum(ERIG)and the problem of HRIG's pathogen pollution can not be ignored.Monoclonal antibody has the potential to replace ERIG and HRIG,but the expression level for eukaryotic system is low resulting in high cost.Therefore,development of engineering antibody y to replace HRIG,ERIG and monoclonal antibody has been very urgent.Single chain variable fragment(scFv)is one of the most studied small molecular antibody,which is composed of connecting peptide antibody heavy chain variable region(VH)and light chain variable region(VL)connected by a peptide linker.ScFv is the smallest unit remaining antigen binding domain.It has several advantages such as small molecule,short half-life,high level of expression and easy operation.Chen Xiaoxu in Jilin University designed scFv98N which is prepared against rabies virus glycoprotein,but the following two shortcomings prevented its further application.First disadvantage is the level of animal protection rate is far lower than that of its parent monoclonal antibody.The second is that the C-terminal of scFv98N has His tag and His tag on the human body are harmful controversial,so clinical research products are requested not to take His tag.For the first problem,through the literature we found that the replacement of VH and VL positions may improve the protection rate of single chain antibody,so according to the scFv98H(VH-linker-VL)sequences,we constructed scFv98R which is a new single chain antibody(VL-linker-VH)to improve its protection rate.According to the second disadvantage,we get rid of the His tag of scFv98H C-terminal and get two new antibodies scFv98N and scFv98RN.With RFFIT protection test in mice and comparing these four kinds of single chain antibody neutralizing activity on the cellular level and animal protection rate,scFv98N is best,and it has no HIS tag,which is more consistent with Pharmacopoeia requirements.This paper designs are mainly as follows:(1)We construct prokaryotic expression plasmid of scFv98R and prepared the two proteins,then compare with the existing scFv98H and scFv98N.Through neutralization potency in the cellular level determined by RFFIT method and protection rate in mice challenged by lethal dose of rabies virus,we found scFv98N is the best,and the scFv98N has no His tag,which is more consistent with Pharmacopoeia requirements.(2)The research of scFv98N separation and purification and optimize conditions.The Qxl was used as the main medium for the purification of scFv98N.the pH,the carrying capacity and the various conditions of the sample velocity were also optimized and separation from the monomer and the polymer.(3)study on the refolding process of scFv98N and optimize its conditions.There are two ways to refold scFv98N.One kind is the column refolding process,but after researching three kinds of chromatography media(Qx1,SP,CM),the column refolding process doesn't achieve a good effect.So dialysis is the main complex method of this experiment.Optimization of initial concentration,pH and other conditions.(4)through the above research to determine the scFv98N purification and refolding of process route,according to the route of purification and renaturation,purity the obtained samples were identified and analyzed by SDS-PAGE and recovery rate was calculated.Western blot detect that if scFv98N has the antigenicity or not.AB SCIEX TOF/TOF is used to measure the molecular weight of the sample,the neutralizing activity of scFv98N is tested by the rapid fluorescent focus inhibition test(RFFIT).Finally,the residual amount of bacterial endotoxin and exogenous DNA were detected. |
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