Preparation Of Monoclonal Antibody Against PLI? And Mechanism Of Anti-inflammatory Effect Of PLI?

Background and objective:Secretory Phospholipase A₂?sPLA₂??E.C.3.1.1.4?is a hydrolase that catalyzes the sn-2 acyl groups on phospholipid molecules.It is also a rate-limiting enzyme to produce inflammatory mediators,such as arachidonic acid?AA?,prostaglandin?PG?,leukotriene?LT?and platelet-activating factor?PAF?,which plays a key role in inflammatory response.PLI??PLA₂ inhibitor gamma?is an endogenous inhibitor of snake venom PLA_2s expressed by snake liver,plays a protective role against self-toxication.In our preliminary study,mammal sPLA_2s are found high identical with snake venom PLA_2s in tertiary structure,thus the venom neutralization protein PLI?is believed also inhibitory to mammal sPLA₂ catalytic activities.In order to verify the their interaction,and investigate the anti-inflammation effect of PLI?,an anti-PLI?monoclonal antibody wasraised on rabbit by our laboratory,and recombinant His6-PLI?was expressed in 293T cell strain to fulfil a in vivo binding validation and subtype identification of sPLA_2s using co-immunoprecipitation and western blot.We have replicated a mouse toe swelling mode by carrageenan inoculation,PLI?of Sinonatrix annularis was administrated by intraperitoneal injection.The anti-inflammatory effects were valued on pathological change by tissue section and HE staining,the sPLA₂ level detected by western blot,the TNF-?and IL-1?expression of toe tissue.These investigation laid the foundation for the drug development of anti-venom and anti-inflammation.This study is divided into three chapters,the main methods and results are listed below:Part 1:Preparation and Identification of Monoclonal Antibody Against PLI?.Methods:1.The PLI?secondary structure,hydrophobic and antigenic epitopes were analyzed by DNAstar software,and the target of PLI?was avoided.Finally,the candidate antigen peptide 151CPVLRLSNRTHEANRNDLIKVA172 was obtained.Named SHE,solid phase synthesis and purification by HPLC.2.SHE and KLH,BSA were conjugated to prepare immunogen and coating antigen.3.After 60?g SHE-KLH conjugates Freund's complete adjuvant and protein emulsified with an equal volume,subcutaneous injection at multiple sites 6-week-old female BALB/c mice.At 14,28 and 42 are respectively injected subcutaneously 30?g of incomplete Freund's adjuvant emulsified SHE-KLH conjugate,for a total of four immunizations.Seven days after the last immunization,the serum titer was measured by iELISA.Titer assay a 96-well plate was coated with SHE-BSA fusion protein.4.After the titer of serum was obtained,the spleen cells of mice and SP2/0myeloma cells were fused with polyethylene glycol?PEG?method,and the hybridoma cells were screened with the complete medium containing HAT.In the same way,ELISA was used to screen hybridoma cells by SHE-BSA.The hybridoma cell line subtype was identified using a mouse immunoglobulin kit.5.Identification of positive hybridoma antibodies.Western blot was performed by prokaryotic expression of His6-PLI?and PLI?.The positive supernatant was detected as primary antibody and His6 antibody was used as positive control.6.Hybridoma cells were expanded into the abdominal cavity of mice,then the ascites was collected and purified by Protein G Sepharose affinity chromatography.7.Western Blot detection of 12 kinds of snake PLI?serum protein,the purification PLI?antibody as the primary antibody.Results:1.Using DNAStar Protean program to predict the secondary structure,flexible region,hydrophilicity,surface accessibility and antigenicity of PLI?.The best hydrophilicity and antigen index were found at 8-27,68-84,108-122,131-136,151-172 and 176-182,and comprehensive secondary structure,surface accessibility to determine the best candidate peptide:151CPVLRLSNRTHEANRNDLIKVA172.The purity of the SHE was 95%by HPLC.2.Twenty-one positive cell lines were obtained by two rounds of ELISA.18subtypes of IgG positive cell lines were obtained after identification.After Western Blot identification,the antibody expression of these 18 cells were both specific to both the native and recombinant PLI?,and the His6-tag antibody same band position.3.Eleven specimen showed bands on the film except Bungarus fasciatus.Interestingly,the molecular weight of PLI?in venomous species were usually higher than that of non-venomous snakes.Part 2:Study on the Interaction between PLI?and Human sPLA₂ Subtypes.Methods:1.The plasmid pIRES₂-EGFP-PLI?was transfected into 293T cells with LipoFiter^TM.Transfection efficiency was observed by fluorescence microscopy at 24and 48 h after transfection.2.The total protein of 293T cells was extracted at 48h,and the interaction between PLI?and sPLA₂ subtypes was detected by immunoprecipitation.Results:1.After transfection for 24h and 48h,green fluorescent protein was observed under fluorescence microscope,and the transfection efficiency was as high as 90%.2.CO-immunoprecipitation assay showed that PLI?interacted with subtypes of sPLA₂ IB,II A,V,X subtypes.Part 3:Study on Anti-inflammatory Effect and Mechanism of PLI?on Mouse Tooth SwellingMethod:1.Balb/c mice were randomly divided into six groups?n=5?:negative control group?NS?,model group,positive control group?2.5mg/kg dexamethasone DXM?,PLI?low dose group?1mg/Kg?,PLI?medium dose group?2.5mg/kg?,PLI?high dose group?5mg/kg?.Negative control group?NS?were injected subcutaneously with30?L saline,and the other five groups were injected with 30?L normal saline in the left hind limb.30?L 1%carrageenan was injected into the right hind limb to construct the toe swelling model.After 30 min injection,the negative control group and the model group were injected with 150?L saline intraperitoneally.The experimental group were injected with an equal volume of dexamethasone?2.5mg/kg?and three doses of PLI?.Before injection of carrageenan,the thickness of the right hind limb was measured using a vernier caliper as 0h as a normal control group.After injection of carrageenan,the thickness of the toes was measured every 1h,and data were recorded continuously for 4h.Calculate the degree of swelling and swelling of the toes in mice.At the 4 hour,the left and right hind limbs of the mice were photographed.The mice were sacrificed and the whole right hind limbs were removed.The rats were sacrificed on the ice.The muscle tissues were removed and the histopathological features of the toes were observed by HE staining.2.Western Blot was used to detect the contents of sPLA₂ IB,II A,V and X in the toe tissue of each group.3.The PLI?protein was incubated with the whole model of the toe total protein at 4°C overnight,and the interaction between PLI?and mouse toe sPLA₂ protein was detected by COIP.4.The levels of TNF-?and IL-1?in the lysates of mice were detected by ELISA.Results:1.Mouse dexticular swollen double hind legs showed that dexamethasone and PLI?could alleviate carrageenan-induced swelling of the toes and PLI?in a dose-dependent manner.There was no significant difference between PLI?high dose group and normal saline group.2.Pathological sections toe mice showed that the model set of slices in a large number of serious neutrophil infiltration and tissue damage.Dexamethasone and PLI?had some effect on neutrophil count and muscle tissue damage.PLI?group was dose-dependent.3.Western Blot showed that PLI?could inhibit the expression of sPLA₂ IB,II A,V and X protein in the toe tissue and had a dose effect,which was more significant for sPLA₂ II A.4.In vitro immune co precipitation verified the interaction between PLI?and sPLA₂ I B,sPLA₂ II A and the inhibitory activity.5.The results of ELISA showed that PLI?had a significant inhibitory effect on TNF-?and IL-1?in mouse toe lysates.Conclusions:1.The PLI?monoclonal antibody and 18 IgG subtype positive cells were successfully prepared.2.The direct interaction between PLI?and sPLA₂ was detected by CO-immunoprecipitation.3.PLI?showed dose-dependent inhibition of carrageenan induced paw edema in mice,significantly reduced the expression of TNF-?and IL-1?of toe Lysates.For its anti-inflammatory action by inhibiting the activity of sPLA₂ II A.