| The West Nile virus disease was caused by the West Nile virus result in an acute infectious disease spread by mosquitoes infection,the main clinical symptoms were included skin rash,fever,Lymph node enlargement,encephalitis and so on.WNV is mainly prevalent in west and central Africa,the Middle East,North America,Asia,Europe,Oceania or the other regions,and there is a huge threat to the public health security.So far the West Nile virus disease were frequently outbreaks in humans and animals,which has brought great economic losses and health threat in the area of high incidence,the West Nile virus disease has been rated as one of the major global epidemic by the Office International Des Epizooties and the World Health Organization.Although there was not nave an outbreak of West Nile virus disease in China,but in 2011 the West Nile virus have been isolated from mosquitoes body by scientific researchers in Xinjiang uygur autonomous region,and the WNV antibody was also detected from sudden encephalitis patients in cerebrospinal fluid.Owing to the frequently trade and tourism in recent years,which had lead to West Nile virus disease had a significantly increased in risk of incoming into China,up to now there have not developed any vaccines or drugs with special effects which can control this disease at present,therefore,it will be having a great significance to research and prepared for a kind of preparations which can effectively prevent and treat the West Nile virus disease.Of this experiment is to study and preparation for the refined equine immunoglobulin F（ab’）2against West Nile virus,the first step were to obtained high immune equine serum against West Nile virus,apply the indirect ELISA detection method of West Nile virus specific antibody which our laboratory has been constructed to monitor the variation of specific antibody titers of high immune equine serum,then take the preparation of high equine serum against West Nile virus by use the saturated ammonium sulfate precipitation method to prepare the high purity of anti-horse Ig G,then digestion the preparation of anti-horse Ig G by pepsin,next step was successively apply the Protein-A column and Protein-G column affinity chromatography to separate and purify the enzyme-digested products,finally get the preparation of high purity refined equine immunoglobulin F（ab’）2 against West Nile virus,next evaluation the preparation of equine refined immunoglobulin F（ab’）2 against West Nile virus in vitro and vivo.Results showed:1)The specificity antibidy titers of the preparation of high immune equine serum against West Nile virus were higher than that of 1:20480.2)The results of SDS-PAGE analysis showed: the preparation of Ig G and F（ab’）2 throughseparation and purification both have good integrity.Ig G shows two complete protein stripe size in about 55 KDa and 25 KDa,F（ab’）2 shows one complete protein stripe size about 25 KDa.Application of thin layer scanning detected,the purity of refined immunoglobulin F（ab’）2successively obtained by Protein-A column and Protein-G column affinity chromatography purification was higher than 90%.3)The results of evaluation in vitro showed: in 80 PFU WNV for testing standards,the calculating results of EC50 were 4.1μg/m L of IgG and 16.5μg/m L of F（ab’）2 respectively.The experimental results show that the preparation of Ig G and F（ab’）2 against WNV has good activity in vitro.4)The results of evaluation in vivo showed: in the condition of infected with 100 PFU WNV in the footpad（s.c.）,and 500μg of the purified equine immune IgG or the purified equine immune F（ab’）2 was injected（i.p.）into infected mice one day before or after infection,or on both days,the mice were monitored for lethality and morbidity daily until 21 days.The experimental results prove that the preparation of Ig G and F（ab’）2 has the good neutralizing ability agsinst West Nile virus in vivo.In the condition of infected with 100 PFU WNV in the footpad（s.c.）,and 500μg of the purified equine immune Ig G or the purified equine immune F（ab’）2 was injected（i.p.）into infected mice one day after infection.After the challenge of mice in day4、day6、day8 and then obtained the spleen and brain tissue from mice respectively in order to detection of West Nile virus load.From the point of treatment effect conditions that immune once of Ig G and F（ab’）2,the treatment experimental results was ideal in vivo.While considering when used for the therapeutic purposes that the Ig G or F（ab’）2 will need to immune many times,therefore we can expected,when under the condition of multiple dosing the therapeutic effect of the preparation IgG or F（ab’）2 will be highlighted（especially aim at the virus removal in spleen and brain）.In conclusion:1)The immune equine serum against West Nile virus was successfully achieved add up to30000 m L;2)The refined equine immunoglobulin F（ab’）2 against West Nile virus was successfullly achieved add up to 1000mg;3)Evaluation results shows that the preparation of refined equine immunoglobulin F（ab’）2against West Nile virus has a good therapeutic effect in vivo and in vitro. |
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