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Screened the Anti-CPV Phage Library and Expressed and Research the Protein VHH

Author: ZhanJunJie
Tutor: DuanZhiBian
School: Shanxi Agricultural University
Course: Clinical Veterinary Medicine
Keywords: Phage antibody library Single domain antibody Biological activity
CLC: S858.292
Type: Master's thesis
Year: 2013
Downloads: 90
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Abstract

Canine parvovirus is a single DNA virus, its structural protein VP2has antigen epitomes which can neutralization antibody. CPV can induce dogs suffering from canine parvovirus disease, that is a highly contagious infectious disease can spread by contacted. Infection rate will reach to100%, if cannot diagnosis and separation the ill Dogs timely.Canine parvovirus disease caused the dog a serious enteritis and myocarditis. At present,the disease has been widely popular all over the country is the most serious infectious diseases harm kennel industry,the average of mortality rate about50%. Now the treatment of the disease, veterinarian used to combined the symptomatic treatment, support treatment and the specificity treatment simultaneously. In the first two treatments, mainly to stop bleeding, anti-nausea, ant diarrheal and fluid infusion, Can only relieve symptoms, but not eliminate the virus. The specificity treatment, most time used high serum and canine parvovirus monoclonal antibodies, but the titer of high serum is not stable and the preparation of monoclonal antibody has a complex process and contained heterogonous protein response, have not effective treatment of the disease, and restricted the clinical application.Part of the alpaca antibodies is heavy chain antibody, using heavy chain antibody cloning VHH which only have heavy chain antibody variable region, VHH has many advantages:good solubility, high affinity, strong specificity, law immunogenicity, small molecular weight (15KD), high stability, and at the same time keep the complete antigen affinity. Build the single domain antibodies gene library to canine parvovirus VP2protein by using the phage display technology. Screened for high expression quantity, strong specificity single domain antibodies, providing a new method for the treatment of canine parvovirus disease.This experiment mainly research contents and results are as follows:1%VHH screened from phage antibody library This prophase experiment construction the phage antibody library to canine parvovirus VP2protein and expression and purification of canine parvovirus viruses VP2protein have already completed. In my study, using the purified VP2protein closed the96-well plate, then after joining activation of canine parvovirus phage antibody library, let the specific phagemid combined with VP2protein. After that competitive eluted with glycine. Used five rounds "adsorption-elution-amplification" enrichment methods for enrichment and screening phage antibody library, meanwhile PCR testing the positive rate of antibody library. Result:original Phage antibody library capacity is1.8×107cfu, after activation is3.6×1011cfu, the first time enrichment7.8×1011fu, the second time enrichment1.07×1012cfu, the third time enrichmentis3.24×1012cfu, thefourthtimeenrichment9.8×1013cfu, and thefifthtimeenrichment3.1×1015cfu. After five rounds screening, the phage antibody library capacity has increased by nearly4000times, PCR results showed that positive rate as high as92%. The single domain antibodies library screening is successful.2、The expression of single domain antibody(VHH) Extract the Phage carrier after a certain amount of amplification,and transferred recombinant plasmid pCANTAB-5E-VHH into expressing host E.Coil HB2151, induced by1mmol·L-1IPTG. PCR and Double enzyme digesting were used to test whether the phagemid vectors containing purpose fragment transferred bacteria. Extraction target protein for SDS-PAGE electrophoresis, to test whether has specific band detection. Result:in PCR experiment there are about400bp size stripe, plasmid and plasmid double enzyme digesting test showed that complete plasmid size is around5000bp, after enzyme digestion there were two stripes, one4500bp,other400bp, the experimental results is consistent with design. Protein electrophoresis results show that there is a band in around14.3KD, there was a specificity protein expressed.3.. Detection the activity of VHH Concentrated on the expressed and specific proteins, using the protein expressed pCANTAB-5E carrier has the characteristics of E-Tag label, we design primary antibody is Anti-E-Tag source form rabbit, second antibody is sheep anti rabbit IgG-HRP for western-blot detection. And then to diluted in different levels of antibodies and antigen, experiment with ELISA to detect the sensitivity and the activity of VHH. Result:western blot experiments showed at around14.3KD has obvious stripe,elisa experiments showed VHH can bind with VP2protein in the concentration of15ug/ml, Suggests that VHH have higher binding activity.VP2protein can be detect in the concentration of250ng/ml, reflect VHH protein has high sensitivity to canine parvovirus VP2protein. When be used for treatment of canine parvovirus disease,VHH can reduce mortality of20%, increase the cure rate of20%, short the treatment period, save the cost10%～20%each one sick dog.

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