The Recombinant Preparation And Detection Methods Study Of Anthrax Toxin Lethal Factor

Anthrax, a fulminating infectious disease of major health importance, is causedby the bacterium Bacillus anthracis. B. anthracis could form spores, which are withstrong survival ability and easy to prepare and spread, under conditions that are notconducive to growth and multiplication of the vegetative forms. That is why B.anthracis has always been high on the list of potential agents with respect tobiological warfare and bioterrorism.The two principal virulence factors of B. anthracis are the toxin complex and thepolypeptide capsule. The function of the capsule in the pathogenesis is protecting thebacterium from phagocytosis, while the toxin is considered as the main harmful agent.Anthrax toxin consists of three components, edema factor （EF）, lethal factor （LF） andprotective antigen （PA）. PA can bind with its cell receptor and form heptamer oroctamer after cleavage by furin-like protease; EF and LF can bind with PA multimer,which lead to a complex that could enter the cell through endocytosis. After being incytoplasm, EF can act as adenyl cyclase and increase the concentration of cAMP,while LF can act as metalloprotease and cleave the N-termini of MEK familymembers. Though the detailed mechanism still remain unclear, it is certain that lethaltoxin （LT, LF and PA） and edema toxin （ET, EF and PA） play prominent roles in thepathogenesis of anthrax and that is why antibiotic treatment can not cure patients inthe late stage of anthrax infection.Accordingly, it is very necessary to detect anthrax toxin accurately: in the earlystage of infection, the detection of anthrax toxin can serve as a diagnosis method;during therapy, real-time monitoring of the toxin concentration is of muchsignificance to evaluate the therapy effect and patient state.LF is an important component of anthrax toxin, and plays an important role ininjury and lethality. Consequently, the establishment of LF detection methods is acapital goal of this study. In order to acquire necessary material, and provide supportsfor other researchers, a simple and efficient preparation process of LF is another goalof this study. At present, key issues in LF preparation are biosafety considerations, nativeamino acid sequence, simple and efficient preparation processes, etc.This study is based on Escherichia coli expression system, which decreases thebiosafety risk. However, we found that the expression level of LF becomes a problem.This study analyzed the expression of LF at the nucleotide sequence level.Through a group of truncated LF mutations constructed and analyzed, and with theaid of a net tool JCat, we presumed that the latter part of the sequence, where tworegions rich of rare codons exist, limited LF expression. After rare codons optimized,the LF expression level indeed got improved.Sequently, we optimized the extract method of periplasm proteins andchromatography workflow. As a result, through osmotic shock treatment and atwo-step chromatography workflow, using a CHT^TMceramic hydroxyapatite columnand a Source30Q anion-exchange column, we were able to obtain sufficient LF（about5mg/L） with high purity （>95%）.The LF preparation was identifized through SDS-PAGE, western blot analysis,N-terminus sequencing, cytotoxicity assay, and rat lethality test. The result indicatedthat this preparation method could provide LF with high purity, native amino acidsequence and full activity, which could not only promote drug and vaccinedevelopment against anthrax, but also facilitate mechanistic research regardinganthrax pathogenesis.After LF preparation process was established, we explored detection methods forLF.Considering that LF is a kind of severe cytotoxic protein, we planed to developea cytotoxicity-based method for detection of LF in the blood plasma. Mousemacrophage J774A.1cell, which is very sensitive to anthrax lethal toxin, was used asan indicator to detect LF in blood. Detection conditions were optimized and acytotoxicity-based method, which can detect as low as5ng/mL LF in the plasma, wasestablished. Method verification was carried out with the samples from Fischer344rats injected with LF, and the half life period of LF in the blood of rats was estimatedat4.8-6.5h.In order to establish a more sensitive and stabilized detection method, wefocused on enzyme activity of LF. In cytoplasm, LF can act as a Zn²⁺-dependentmetalloprotease, and cleave members of MEK family. At present, several detectionmethod researches have targeted on this character. However, most of them referred mass spectra or high performance liquid chromatogram, which complicated thedetection processes.Herein, we constructed a recombinant plasmid, which contained coding genes ofa His tag, the LF cleavage site in MEK, and the E. coli alkaline phosphatase. Theexpressed fussion protein （MEKAP） was used to detect LF in a simple process likeenzyme linked immunosorbent assay. Thus, without mass spectra or high performanceliquid chromatogram equipment, we could detect LF with a fine sensitivity.In summary, this study established a simple and efficient LF preparation processfirst, and then explored detection methods for LF. A cytotoxicity-based LF detectionmethod was established and verified with the samples from Fischer344rats injectedwith LF, and the half life period of LF in the blood of rats was estimated. A simpledetection method targeting on LF enzyme activity was designed and proved to bepracticable. These results facilitated clinical diagnosis and therapy and mechanisticresearch regarding anthrax.