The Preparation Of LINGO-1 Polyclonal Antibody And Experimental Study Of Its Passive Immunotherapy For Acute Spinal Cord Injury In Adult Rats

BackgroundThe self-repair function of nerve tissue is extremely limited after spinal cord injury, largely due to the micro-environment is not conductive to axon regeneration. It is mainly formed by many axon growth inhibitor released from the collapsing tissue. Recent data indicate that many different axon growth inhibitors could bind the neuronal membrane NgR receptor complex(NgR/p75/LINGO-1 or NgR/TAJ (TROY)/LINGO-1),activate intracellular RhoA signal,and then result in growth cone degeneration and axon regeneration failure.LINGO-1(LRR and Ig domain-containing,Nogo Receptor-interacting protein,LINGO-1) is an important component of the NgR receptor complex,and also present in oligodendrocytes.It functions as a negative regulator of oligodendrocyte differentiation and myelination, neuronal survival and axonal regeneration. The targeted inhibition of LINGO-1 therefore presents a novel therapeutic approach for the treatment of neurological diseases.Some researchers immunized SCI animals with inhibitor proteins, leading to the production of neutralization antibodys which can block the effect of inhibitors, improve the local micro-environment and promote axonal regeneration and function recovery.But it takes about 6-8 weeks for neutralization antibody production when immunized immediately after injury.It is more adapted to explore means of passive immunization to promote early functional recovery of SCI.We suppose that the useage of LINGO-1 polyclonal antibody can block the function of LINGO-1,weaken the downstream inhibitory signal,decrease the neuronal apoptosis and promote axonal regeneration and functional recovery. The primary option of antibody therapy is monoclonal antibody for its best specificity and quality control.But It will take much more time and higher ecnomic input for preparation.In addition,the first step for antibody therapy is to find passive immunization of antibody therapy can block the targeted candidate and harvest functional recovery after SCI.For these purposes,we prepared a recombinant human LINGO-1 protein as immunogen and producted a large amount of polyclonal antisera of high titer and good specificity for LINGO-1.This study planed to explore the intervention of LINGO-1 antisera to spinal cord dorsal hemisection in adult rat,expect to provide new idea for SCI treatment.The reseach is divided into three parts.The first one is the preparation of LINGO-1 polyclonal antisera;the second one is to explore the feasibility of LINGO-1 polyclonal antisera treatment With spinal cord injury model in adult rats;the last is to observe whether the polyclonal antibody can promote the axonal regeneration and function recovery of the animal model.Part 1 The preparation and identification of LINGO-1 polyclonal antibodyObejectiveTo express and purify the LINGO-1 ectodomain fusion protein with poly- histidine tag,then to prepare the rabbit-derived anti-hLINGO-1 polyclonal antisera.Methods(1) PCR amplification of LINGO-1 of 76-319 amino acid coding sequence cDNA To design specific primers according to the cDNA sequence hLINGO-1aa76-319,introduce BamH I, EcoR I restriction sites and the protection bases on the 5'end of upstream and downstream primer respectively.Plasmid pCMV-SPORT6-LINGO-1 as a template,to amplificate hLINGO-1 cDNA fragment under high-fidelity Taq enzyme by PCR.(2) Construction of Prokaryotic expression vector pET30a(+)-hLINGO-1aa76-319 To treat recycling purified PCR product and vector with BamH I, EcoR I double digestion,recycle and ligate the target gene pieces,then transformate DH5a feel-state bacteria.Pick out monoclonal colonies, shake bacteria, extract plasmid. Identify the recombinant plasmid by PCR, double enzyme digestion and sequencing.Sequences thus obtained were compared to GenBank database using BLAST program,then the validated gene fragments were inserted into pET30a (+) expression vector.(3) the optimal conditions of hLINGO-1aa76-319 fusion protein induced expression To transform pET30a(+)-hLINGO-1aa76-319 recombinant plasmid into BL21 feel-state bacteria,pick out the positive monoclonal colonies by PCR identification, inoculate in kanamycin-resistant LB medium.Add different final concentration of IPTG for induction,when the OD is between 0.6-0.8.Collect samples at different time-points for SDS-PAGE electrophoresis,then staining by Coomassie brilliant blue for view the protein bands,and determine the optimal induction condition.Centrifuge the bacilli which contain highest amount of protein expression and re-suspended sedimentation by split buffer, get the Supernatant and sedimentation by ice bath ultrasonic lysis, then perform SDS-PAGE and western blotting to verify the expression form of fusion protein.(4) Purification of fusion protein after substantial expression Cultivate 1000mL bacilli in optimized expression conditions, get sedimentation by Centrifuge the bacilli and re-suspending by split buffer,harvest inclsion bodys by ice bath ultrasonic lysis and centrifugalization.resolve inclsion bodys in urea and recycle the LINGO-1 fusion protein by chelating resin chromatography.The concentration of purified protein was determined by BCA protein assay.The protein pufity was determined by Bandscan 5.0 software.For higher purity protein antigen,all the fusion protein was loaded on the SDS-PAGE electrophoresis.The acrylamide piece with antigen were cut out and preserved at-80℃.(5) The preparation of hLINGO-1aa76-319 antisera After frozen overnight,glue block grind with Freund's adjuvant adequately.After full emulsification, multi-point injection was done at rabbits back. strengthen the immune response for every 7 days.Harvest sera before immunization for negative control and a small amount of boold 10 days after 3rd immunization. antisera titer was detected by ELISA.When the titer achieved the desired level,collect the blood for antisera preparation and preserve at -80℃.(6) Identification of antisera specificity and titer detection the total protein of BL21 expressing the fusion protein and brain membrane protein of rat were perform by SDS-PAGE electrophoresis,then the prepared antisera for the first antibody to verify its speciality by western blotting.The ELISA plates were coated overnight at 4℃with 4μg/ml LINGO-1 fusion protein. While sera before immunization for negative control,the antisera titer was detected by ELISA.Results:(1) PCR amplification of LINGO-1 of 76-319 amino acid coding sequence cDNA one clear band could be seen around 732bp under UV light after agarose gel electrophoresis,is as the same length as the purpose.(2) Construction of Prokaryotic expression vector pET30a(+)-hLINGO-laa76-319 Double-digested hLINGO-laa76-319 PCR products were insert into the prokaryotic expression plasmid pET30a(+) and Extract the screening recombinant plasmid DNA.732bp fragment was got by PCR amplification. 5.4kbp and 732bp fragment were got by BamH I and EcoR I double-digested identification.They were coincident with the expect.The sequencing result was the same as the sequence provided by GeneBank(serial number:NM032808). These results verified that the vector was constructed sucessfully and it was named pET30a(+)-hLINGO-laa76-319.(3) The optimal conditions of hLINGO-laa76-319 fusion protein induced expression Bacteria BL21 transformed recombinant plasmid,identified by PCR,was inducted by IPTG at different conditions.Collect the total Bacterial protein for SDS-PAGE electrophoresis.The result indicated the fusion protein have highest expression at 37℃,0.4mM IPTG induction for 1.5 h.Then the fusion protein was be confirmed by western blotting with anti-His tag mAb for the first antibody. Bacterial precipitation broken by ultrasonic,the suprenant and sediment were collected for SDS-PAGE electrophoresis.The result confirmed that the fusion protein was in the form of inclusion body.(4) Purification of fusion protein after substantial expression Expanse culture system according to the optimal condition of step three, harvest inclusion bodys, then recycle the fusion protein after purification.The concentration of protein was 4600 mg/L determining by BCA assay,and the purity was 70.8%.(5) The preparation of hLINGO-laa76-319 antisera Control sera was harvested before immunization by repeated blood sample method.Rabbits have Normal feeding,be in good health and no significant adverse reactions in whole immune process.serum titers were higher than 1:312500 after 3rd immunization. after fourth immunization,The volumn of blood harvested was 197 ml,and the volumn of prepared antisera was 61 ml.The control sera and antisera were filtered by 0.22 um filter membrane,preserved at-80℃.(6) Identification of antisera specificity and titer detection The titer of harvested antisera was 1:1562500. the total protein of BL21 expressing the fusion protein and brain membrane protein of rat were perform by western blotting,and the prepared antisera was be the first antibody.The colored protein band was the expected result,these results reprsent that the prepared antisera has high titer and good specificity.ConclusionsThe prepared hLINGO-1aa76-319 antisera has good specificity and high titer,and has laid an important foundation to further study the biological function of LINGO-1 protein.Part 2 The permeability and specificity of LINGO-1 Polyclonal Antibody in vivoObjectiveTo analyzed the permeablilty and specificity to the antigen of LINGO-1 polyclonal antibody which administered locally into the spinal cord injury site of rats.MethodsTwelve Sprague-Dawley female rats(200-250g) were randomly divided into three groups:normal group, control group(hemisection+control sera) and experiment group (hemisection +antisera).Normal group was without any treatment,the other two groups were performed T9 dorsal hemisection,completely interrupting the main dorsomedial and dorsolateral corticospinal tract(CST) components.Immediately after CST transection,an intrathecal catheter was inserted into the subarachnoid space at T9 and connected to a primed mini-osmotic pump inserted into the subcutaneous space.Mini-osmotic pumps delivered control sera and LINGO-1 antisera respectively. 3 d and 28 d after hemisection,each T8-T10 segments of each group were harvested for cryosection and immunofluorescence tissue staining,statistical analysis was done by two independent samples t test,to analyzed whether LINGO-1 polyclonal antibody can penetrate into spinal cord tissue and bind with LINGO-1 molecule specifically.ResultsRat bilateral lower limbs were completely paralyzed and low muscle tone when awaked from operation.This proved that the model-making was sucessful.Three days after operation,spinal cord injury sites of normal group could not detect rabbit-derived antibody,control group and experiment group could detect penetrating antibody which come from rabbit.Twenty-eight days after operation,the result was the same as before.Three days after operation,Mean immunofluorescence density of experiment group was significantly lower than control group(P<0.05),and Mean immunofluorescence density of control group,which section pre-treated with LINGO-1 antisera,was also significantly lower than control group which pre-treated with rabbit-derived control antisera(P<0.05).ConclusionsThe spinal cord dorsal hemisection model was successfully set up,which has laid an important foundation to further study the effectiveness of antisera treatment. LINGO-1 polyclonal antisera,administered locally,can detected at the spinal cord injury site in wide time-window and recognized LINGO-1 molecule specifically.It is feasible that the method of passive immuno-therapy for spinal cord injury.Part 3 Efficacy assessment of LINGO-1 polyclonal antibody on spinal cord hemisection model in ratsObjectiveTo assess the efficacy of LINGO-1 polyclonal antisera administered locally on spinal cord hemisection model in rats.MethodsFourty-nine Sprague-Dawley female rats(200-250g) were randomly divided into three groups:normal group(n=3), control group(hemisection+control sera, n=23) and experiment group (hemisection +antisera,n=23).Normal group was without any treatment,the other two groups were performed T9 dorsal hemisection,completely interrupting the main dorsomedial and dorsolateral corticospinal tract(CST) components.Immediately after CST transection,an intrathecal catheter was inserted into the subarachnoid space at T9 and connected to a primed mini-osmotic pump inserted into the subcutaneous space.Mini-osmotic pumps delivered control sera and LINGO-1 antisera respectively.Three days after hemisection,active RhoA of spinal cord tissues were assayed by protein pull-down technology and western blotting, apoptotic neuron death of each group was assessed by Immunofluorescence double-standard method.Fourteen days after hemisection,animals were re-anesthetized and the right sensory-motor cortex exposed via a craniotomy and biotin dextran amine(BDA) in PBS, stereotaxically injected at the motor cotex to label the CST axon.Twenty-eight days after hemisection, animals were killed to collect injury site tissues for cross-section and longitudinal section.BDA Immunohistochemistry staining was taken for tracing CST.Animals were scored using the open field BBB scoring system.Rats were evaluated the day after CST transection(day 2) and weekly thereafter for 4 weeks with observers blinded to the treatment regimen. Quantification of the active RhoA protein was statistically analyzed using the One-Way ANOVA and followed by a post hoc LSD test.Quantification of apoptotic neurons was analyzed by independent-samples t test.The maximum regenerating length was analyzed by two independent sample tests.BBB behavioral scores was analyzed by repeated measures ANOVA followed by independent-samples t test.Differences were considered to be statistically significant when P<0.05.Results(1) In active RhoA assay experiment,the RhoA total expression in each group has no difference between each other.But the highest expression of active RhoA is control group,followed by the experimental group,the normal group at least.Acquired the active RhoA densitometry index(K) in each group was for statistics,the total RhoA protein of each group was to be the calibration standards.Accrording to ANOVA analysis,there were significant difference in inter-groups(F=686.711, P=0.000). Then accroding to LSD mutiple-comparisons, there were significant difference between groups(P=0.000).The active RhoA in control group was significantly higher than normal group(P<0.001).The active RhoA in experimental group was significantly lower than normal group(P< 0.001),but still higher than normal group(P<0.001).(2) In apoptotic neuron detction,six sections in each sample were be taken for counting in blind manner.the result was taken for two independent sample test. there were significant difference between this two groups(t=2.395, P=0.022).This verified that the number of apopototic neuons in experiment group was significantly less than control group and consistent with the result of active RhoA assay. (3) To evaluate the effect of LINGO-1 antisera on axon regeneration,BDA anterograde tracing was used to label the regenerated CST fibers in both experimental group(n=16) and control (n=15) group. Two weeks after BDA injection, transverse and sagittal sections were collected for BDA staining and the maximum length of labeled fibers, extending caudal to the lesion site, was estimated from serial sections. In transverse sections 11-16 mm rostral to the lesion site, CST fibers were equally labeled in both experimental and control groups.However, in transverse sections 11-16 mm caudal to the lesion site,the labeled fibers could not be detected in control group, while in 25% of experimental group(4 of 16), BDA labeled dorsal axons were still observed in the corresponding area. In longitudinal sections across the lesion site, all of the BDA labeled CST fibers in the control group ceased above or at the lesion sites and the caudal growth of labeled axons was not detected. In contrast, in 75% of experimental group (12of 16), BDA labeled axons sprouted across the lesion area and extended within the caudal part of the spinal cord.The maximum distance of labeled CST axons ranged between 5.7 mm to 8.1 mm distal to the injury(Md=7.9mm).(4) The dorsal and dorsolateral components of CST were completely interrupted and the ventral portion of the CST left intact.Rat bilateral lower limbs were completely paralyzed and low muscle tone when awaked from operation. Hindlimb function was quantified using the Basso-Beattie-Bresnahan (BBB) open field scoring method(Fig.3-3). Control animals receiving continuous intrathecal infusion of control sera recovered substantial function over the 4 week duration of the experiment, attaining a mean BBB score of 12.2±1.5. Continuous intrathecal infusion of LINGO-1 antisera for 4 weeks after spinal cord transection resulted in significantly improved BBB scores at 3 and 4 weeks,reaching a mean BBB score of 14.4±1.4.A BBB score of 14 or greater reflects the ability of rats to display consistent coordinated hindlimb-forelimb movement and represents a threshold of recovery that is biologically meaningful.The frequency with which LINGO-1 antisera-treated animals attained a score of 14 or more was greater than for control animals,3 weeks (37.5% VS 13.3%, P=1.000) and 4 weeks (75.0% VS 20.0%, P=0.003) after SCI. Together, these results clearly demonstrate that treatment with LINGO-1 antisera promotes the recovery of function after SCI.ConlusionsLINGO-1 polyclonal antisera can effectively block the activation of downstream inhibitor signal in spinal cord tissue,reduce neuronal apoptosis, significantly decrease the influence of secondary injury,have the effects of protecting nervous tissue,promoting axonal regeneration and motory function recovery.