Study On Immunoprotective Effect And Mechanism Of The Functional Domain Epitope Of Botulinum Toxin Type A

Botulinum neurotoxins?BoNTs?are toxic proteins secreted during the growth and reproduction of botulinum,and have strong neurotoxicity.According to their antigenic differences,BoNTs can be divided into 7 types of BoNT/A-G,among which A,B,E and F can cause human poisoning,C and D type only cause animal poisoning,and poisoning caused by G type is rare.Among all known toxins,BoNT/A is the most toxic substance,and the lethal dose in adults is:injection of 0.09 to 0.15?g,inhalation of 0.70 to 0.90?g,and oral administration of 70?g.Due to its strong toxicity,simple preparation and convenient transportation,BoNTs is listed as a Class A biological warfare agent internationally and is listed as one of the six most important biological warfare agents.BoNTs have obvious sequence homology,so their structure and function are similar:they consist of a light chain?L,molecular weight 50 kDa?and a heavy chain?H,molecular weight 100 kDa?,which are connected by disulfide bonds.The carboxy terminus of the heavy chain?Hc,molecular weight 50 kDa?is the receptor binding region and is responsible for binding to the neuronal receptor.It is composed of the C-terminus of the receptor binding region?Hc-C,molecular weight 25 kDa?and the N-terminus of the receptor binding region.?Hc-N,molecular weight 25 kDa?consists of two subdomains;the amino terminus of the heavy chain?HN,molecular weight 50 kDa?is the transporter region,responsible for transmembrane transport function;The light chain is the catalytic region,which is the most studied region in the BoNTs domain.The poisoning mechanism of BoNTs is divided into four steps:binding to the recognition of target cell receptors,internalization,transmembrane transport of the catalytic domain and activation of toxins,and finally cleavage of soluble N-ethylmaleimide sensitive molecular attachment protein receptor?SNARE?leads to neurotransmitter release block caused by skeletal muscle paralysis,causes respiratory failure and death.Vaccination is the most effective strategy to prevent botulism.At present,the research on botulism vaccine mainly focuses on novel toxoid vaccine,new recombinant subunit vaccine,nucleic acid vaccine,and the most research prospect is the new recombinant subunit vaccine.The most ideal subunit vaccine should be the smallest antigenic fragment that can significantly cause the body's protective immune response,in a sense,the choice of target antigen determines the specificity,protection and effect of the immune response.Our experiment takes BoNT/A as the object,to study the immune efficacy of epitope antigens in various functional domains,and to explore the mechanism of immune protection by recombinant antigens,and provide basic data and theoretical support for the development of BoNT/A recombinant subunit vaccine.In this study,the functional fragment of BoNT/A functional domain was prepared by gene engineering and ligated into pTIG-Trx prokaryotic expression vector,the constructed plasmid was transformed into E.coli system to induce expression and passed through Ni-NTA affinity chromatography purify provides high purity recombinant antigen?such as Hc,HN-L,etc.?.The immunopotency protection experiment demonstrated that the recombinant Hc and HN-L antigens can produce a good immunoprotective effect after two immunizations,and can completely resist the attack of 10⁴ LD₅₀ BoNT/A,the serum neutralization titer is 6.6 IU/ml,achieving a high protective titer level.When Hc and HN-L antigens were combined,the neutralizing potency increased to 8.8 IU/ml,indicating that the combination of the two can have a synergistic effect.The results of ELISA assay showed that Hc and HN-L antigens could induce specific high-level antibodies after immunization,which showed high levels of humoral immune response,indicating recombinant Hc and HN-L expressed and purified by E.coli system have good immunogenicity.Recombinant Hc-C,Hc-N,and HN antigens also produced certain immunoprotective effects after booster immunization,and the results of neutralization titer reached the protective level of the organism.However,the recombinant L and small fragment sHN-L antigens did not produce immunoprotective effects after immunization,and the neutralization titer was<0.1 IU/ml,and there was no obvious protective effect.In addition,a single dose-dependent immunoprotective study of important antigens was performed.The results showed that the half-effective dose ED₅₀ of 10³ LD₅₀ BoNT/A after single-dose immunization with Hc antigen was about 0.125?g,the half-effective dose ED₅₀0 of 10³ LD₅₀0 BoNT/A after single-dose immunization with HN-L antigen is about 1.215?g,the half-effective dose ED₅₀ of 10³ LD₅₀ BoNT/A after single dose of Hc+HN combined with antigen is about 0.285?g.the half effective dose ED₅₀ of 10³ LD₅₀BoNT/A after single dose immunization with Hc+HN-L combined with antigen was approximately 0.069?g.The results of this single-dose-dependent experiment prove that Hc is still the best target antigen of the toxin subunit vaccine,and its protective effect is equivalent to the immune effect of the full-length toxin,which can be used for the development of the toxin subunit vaccine.Neutralizing epitopes are the core targets of antigen immunization,in order to explore the immunoprotective mechanism of BoNT/A antigenic fragments,this study carried out the study of neutralizing epitopes on recombinant antigen fragments from three aspects:binding site,spatial structure and affinity.Firstly,three specific neutralizing antibodies A1,A2 and A3 of BoNT/A were prepared and tested for neutralizing potency.Th Then,the antigen and the antibody can be specifically combined in vivo or in vitro to determine the ability of the antibody to bind to each recombinant antigen.The results of ELISA and Western Blot binding assay showed that the two antibodies A1 and A2 bind to Hc and Hc-C antigens,do not bind to other antigens,and can bind under denaturing or reducing conditions,indicating that the neutralizing epitope to which they bind is located on Hc-C and is linear epitope;while A3 binds only to the HN-L antigen,does not bind to the sHN-L,HN and L antigens,and does not bind to denatured or reduced HN-L,indicating that it binds to the binding region of HN-L and is conformational epitope.And the detection of antibody affinity by ELISA showed that the three antibodies had a very high affinity with the neutralizing epitope.The above results demonstrate that the Hc and HN-L antigen domains have well-defined neutralizing antibody epitopes,which can induce the production of neutralizing antibodies and protection in the body.In addition,the Hc-C+Hc-N and HN+L combination groups did not produce clear strong immunoprotective effects compared to intact Hc and HN-L antigens,this suggests that neutralizing antibody epitopes require intact structural molecules to induce neutralizing antibodies and protection.In summary,this study completed the preparation and identification of functional epitope epitopes of BoNT/A,and studied the immunoprotective efficacy and immunoprotective mechanism of recombinant antigens.The results showed the Hc and HN-L antigen fragments have the best immunogenicity and immunoprotection,the important neutralizing antibody epitopes are mainly concentrated on the Hc and HN-L antigens,of which Hc has two and HN-L has one.In conclusion,the Hc receptor binding domain molecule has the appropriate size and ease of preparation,and can producs complete immunoprotection,and has superior efficacy over HN and HN-L,and is comparable to Hc+HN-L full-length antigen.Therefore,it can be used as an ideal target antigen for BoNT/A recombinant subunit vaccine,and is used to develop a recombinant toxin vaccine instead of a toxoid vaccine.