Cloning, Expression And Purification Of Yersinia Pestis F1-V Fusion Protein In Escherichia Coli And Studies Immune Protection In BALB/c Mice

Plague is a zoonotic infection that is spread to humans from natural rodent reservoirs, commonly via the bite of an infected flea. The bacteria are normally transmitted to humans from a flea vector. People bited by infected flea of Yersinia pestis could emerge bubonic plague and then septicemic plague and pneumonic plague subsequently. Pneumonic plague is spread via respirtory droplets through close contact in human beings. Before without good effective drugs, people infected pneumonic plague disease are all dead. Without vaccinated people death rate is between 40% to 80% in bubonic plague. Each year there are several thousand reported cases of the disease world-wide and plague has been classified as a re-emerging disease by the World Health Orgnization.The Surat outbreak of plague in India in 1994 reminded the world of the threat of this horrible disease. Plague is an international public health risk and a notifiable disease. More over, Y.pestis has been of concern as one of the microorganisms which might be used illegitimately as a bioterrorisim agent against civilian or military communities. Because of the increasing plague cases and likelihood of illegitimate use of Y.pestis, there is a requirement for an effective vaccine which can protect human against both bubonic and pneumonic plgue.There are two kinds of vaccines have been used in human, live attenuated vaccine and killed whole cells vaccine (KWC). The earliest report of a killed whole cell vaccine against plague in 1897, and USP (a KWC vaccine) has been used in Vietnam duringthe period 1961-1971. But recent studies in animals have shown that this vaccine offers poor protection against pneumonic disease. Another vaccine, live attenuated vaccine (EV76 strain), has been in used since 1908, and ws used in many countries in the late20^th century. However, as EV76 is not fully avirulent, the safety of this vaccine in human is questionable. Today EV76 is considered unsuitable for humans in most countries. The efficacy and the safety of existing vaccines are not proved, and the use of these vaccines is not known to be associated with adverse side effect. Therfore there is an urgent need for a more efficacious plague vaccine. In recent years, efforts have focused on the development of fullyrecombinant subunit vaccine for plague and identification of a rationally attenuated mutant strain of Y.pestis.In recent research demonstrated, immune protection of Y. pestis is mainly determined two parts, one is capsule-like Fraction 1 (F1), it expressed F1 protein by determine 65mD plasmid and encoded molecular mass about 15.5kD polypeptide. Fl protein has pooly tocxity but good immunogenicity, including numerous determinations antigen and its ability to resistance to phagocytosis in vivo. Fl antigen is ability to induce capsule-like antibody and relate with plague infection. V antigen is another protective molecular of Y. pestis and determine by 45mD plasmid, encoded molecular mass about 37kD polypeptide. V antigen has also pooly tocxity but good immunogenicity, including numerous determinations antigen and its ability to resistance to phagocytosis. It can promote bacteria of plague proliferation in monocytes. F1 antigen and V antigen are two major protective antigen and conservation in species. Fl antigen and V antigen have super impose effect in immune protection and immunogenicity. They become first protective molecular in new vaccines research of Y. pestis.In our research, EV76 genome plasmid has extrated from the strain of Yersiniapestis. Fl gene and V gene of Yersinia pestis were cloned and inserted into pGEM-T vector. After corrected sequence, the recombinant expression plasmid F1-V/pET-11c was constructed by inserting the DNA fragment of Y. pestis Fl gene and V gene into pET-11c and transformed into E.coli BL21 (DE3) cell. The positive clones were screnced out by PCR and enzymes digesting and then through IPTG inducement expression, the expressed product was detected by SDS-PAGE.Sequence analysis revealed that Fl gene DNA sequence was completely same as that of Gene-bank record (X61996). DNA sequence of V gene was mutanted at the site of 546bp, but encoded same amino acid. An expression band about 58000 Dal was found by SDS-PAGE.The aim protein representing about 22%of total cell protein and the most in soluble form. While the expression protein immunogenicity was detected by Western-Blot.The aim protein has immunogenicity by against plague.Different purification strategies were used to purify F1-V/pET-11c. First hydrophobic chromatograph was adapted, then anion exchange chromatograph was used and last size exclusion chromatograph was adapted. The purity of Fl-V fusion protein is more than 90% by scaning.An alhydrogel-absorbed vaccine of this Fl-V fusion protein has been intramuscularly injected with BALB/c mice. Immunogenicity was detected and anti-serum titres were detected of F1-V fusion protein by ELISA .The titres of IgG antibody by immunized Fl-V fusion protein with alhydrogel was reached to l:51200±800. T cell prolifiration were detected by MTT. It was able to induce an effective immuno-protection against subcutaneous challenge of virulent Y. pestis with 400 minimal lethal doses (MLD) by intramuscularly injected with three doses into experimental BALB/c mice. The survival rate was 90%. F1-V fusion protein was able to against subcutaneous challenge of...