Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

EXPRESSION CLONING OF A PROTECTIVE LEISHMANIA ANTIGEN

Parasite-specific CD8+ T cells have been shown to transfer protection against nLeishmania major in susceptible BALB/c mice.An epitope-tagged expression library was used to identify the antigen recognized by a protective CD8+ T cells clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histompatibility complex class n. A conserved 36-kilodalton member of the tryptophanaspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12before injection.

Construction and Expression of Prokaryotic Expression Vector of MPT-64 Gene

[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.

Safety,immunogenicity,and cross-species protection of a plasmid DNA encoding Plasmodium falciparum SERA5 polypeptide,microbial epitopes and chemokine genes in mice and olive baboons

Incorporation of biomolecular epitopes to malarial antigens should be explored in the development of straintranscending malarial vaccines.The present study sought to determine safety,immunogenicity and cross-species efficacy of Plasmodium falciparum serine repeat antigen 5 polypeptide co-expressed with epitopes of BacilleCalmette Guerin（BCG）,tetanus toxoid（TT） and a chemokine gene.Olive baboons and BALB/c mice were randomly assigned into vaccine and control groups.The vaccine group animals were primed and boosted twice with pIRES plasmids encoding the SERA5 ＋ BCG ＋ TT alone,or with either CCL5 or CCL20 and the control group with pIRES plasmid vector backbone.Mice and baboons were challenged with P.berghei ANKA and P.knowlesi H strain parasites,respectively.Safety was determined by observing for injection sites reactogenicities,hematology and clinical chemistry.Parasitaemia and survivorship profiles were used to determine cross-species efficacy,and T cell phenotypes,Th1-,Th2-type,T-regulatory immune responses and antibody responses were assessed to determine vaccine immunogenicity.The pSeBCGTT plasmid DNA vaccines were safe and induced Thl-,Th2-type,and Tregulatory responses vaccinated animals showed enhanced CD4~＋（P〈0.01）,CD 8~＋ T cells（P〈 0.001） activation and IgG anti-SE36 antibodies responses（P〈 0.001） at week 4 and 8 post vaccination compared to the control group.Vaccinated mice had a 31.45-68.69%cumulative parasite load reduction and 60%suppression in baboons（P〈0.05）and enhanced survivorship（P〈 0.001） with no clinical signs of malaria compared to the control group.The results showed that the vaccines were safe,immunogenic and conferred partial cross-species protection.