Study On Ip/pilE DNA Vaccine Against Legionella Pneumophila

Legionella pneumophila, the most common cause of Legionaires' disease, is a facultative intracellular parasite bacterium that causes epidemic, sporadic, and nosocomial pneumonia. Epidemiological evidence suggests that the environmental source of theses infection is natural freshwater systems and man-made aquatic environments. Human infection occurs by the inhalation of aerosolized bacteria from L. pneumophila-contaminated water sources. L. pneumophila invades and multiplies within alveolar macrophages. The major outer membrane protein and typeⅣpili are associated with L. pneurnophila infection of macrophages have been reported. In the outer membrane of L. pneumophila, there is a protein with molecular weight of 29 kDa, which is not only an specific antigen of the species but also the major outer membrane protein; it also be factor ofpathogenecity in the bacterium. The 29 kDa protein (29 kDa immunogenic protein, IP) was encoded by the ip gene. The mechanisms still remain unclear. The presence of typeⅣpilin gene (pilE) and its expression by L. pneumophila provide an advantage for colonization of lung tissues and invasion of macrophage during Legionnaires'Disease. According to the current study, an astonishing number of cellular functions have been attributed to the typeⅣpili, including surface motility, microcolony and biofilm formation, adhesion, immune evasion, cell signaling, transformation of DNA and phage attachment. These observations suggest that the 29 kDa protein and typeⅣpili may play a vital role in the host immune response, therefore develope some safe and effective vaccines for the disease will be of practical significance.This study selected Legionella pneumophila DNA vaccine as research direction. According to the results of indexing and analysis in the relevant literature, this study selected ip gene, pilE gene of Legionella pneumophila as candidate genes of the DNA vaccines. We have used the technique of splicing by overlap extension by the polymerase chain resction (SOE by PCR) to produce the ip and pilE fusion gene of Legionella pneumophila. The eukaryofic expression recombinant plasmid containing the ip gene, pilE gene and ip/pilE fusion gene were constructed successfully. And then study the immunity characteristics of these DNA vaccines, which provided certain experimental basis for the study of the new generation of safe and efficient DNA vaccines.The study was divided into six parts.In the first part, the ip gene of 792 bp long was amplified from the total cell DNA of Legionella pneumophila with PCR, and then was inserted it into the prokaryotic expression vector pET-32a(+). After the recombinant plasmid pET-ip was identified. And also the Trx-IP fusion protein of approximately 49 kDa in size was expressed in prokaryotic cell efficiently as expected.In the second part, the pilE gene of Legionella pneumophila was amplified from the genomic DNA ofLegionella pneumophila with PCR, and then was inserted into the prokaryotic expression vector pET-32a(+). The prokaryotic expression recombinant plasmid pET- pilE was constructed. After identified with restriction enzyme analysis and polymerase chain reaction and nucleotide sequencing analysis, the Trx-PILE fusion protein of approximately 35.7 kDa in size was expressed efficiently as expected.In the third part, the fragments of the ip and pilE gene having matching sequences at their ends to be fused were amplified from the genomic DNA of Legionella pneumophila by PCR respectively, and then these PCR products were mixed, denatured, reannealed, the strands with matching sequences at their 3' ends can overlap and serve as primers for each other. Extension of this overlap by DNA polymerase produces recombinant products. After amplification by using outer primers, the ip/pilE fusion gene of about 1218 bp long was amplified and then it was inserted into the prokaryotic expression vector pET-32a(+), the recombinant plasmid pET- ip/pilE was constructed successfully and also the approximately 64.7 kDa Trx-IP-PILE fusion recombinant protein in size was expressed as expected.In the fourth part, the ip gene, pilE gene and ip/pilE fusion gene was amplified by PCR. The products of PCR were inserted respectively into eukaryotic expression plasmid pcDNA3.1(+). The eukaryotic expression recombinant plasmid pcDNA3.1-ip, pcDNA3.1-pilE and pcDNA3.1-ip/pilE were also constructed respectively. NIH3T3 cells were transfected by the recombinant plasmid pcDNA3.1-ip, pcDNA3.1-pilE and pcDNA3.1-ip/pilE with lipofection strategy. Transient products were detected in cell membrane and inside the cell by immunofluorescence. And about 29 kDa protein of the IP, 15.7 kDa protein of the PILE, and IP-PILE fusion protein of approximately 44.7 kDa in size were detected in stable transfercted cells with Westem-blot.In the fifth part, BALB/c female mice were immunized intramuscularly with the naked DNA vaccine, and an blank group was immunized intramuscularly with PBS. Immunogenicity was evaluated through antigen specific antibodies, lymphocyte proliferative response, IFN-γproduction, IL-2 production and cytotoxic T-lymphocyte response of immunized mice. The results showed that immunogenicity of DNA vaccine immunized mice were higher than control group. Humoral immunity and cellular immunity of each group of DNA vaccines immunized mice was higher than pcDNA3.1(+) immunized mice. Antigen specific antibodies of pcDNA3.1-ip/pilE immunized mice were higher than peDNA3.1-pilE immunized mice. Cell mediated immune responses of pcDNA3.1-ip/pilE immunized mice were higher than pcDNA3.1-ip, pcDNA3.1-pilE immunized mice. Immunogenicities of pcDNA3.1-ip immunized mice were higher than pcDNA3.1-pilE immunized mice.In the sixth part, BALB/c female mice were immunized intramuscularly with the DNA vaccines. 2 weeks after the second immunization, the immunized mice were challenged with L.pneumaphila serogroup 1. On the day of infection, bacteria were diluted in normal saline deliver approximately 10 times the 50% lethal dose that consistently resulted in the death of all control mice to each animal. The bacterial counts and the pathological section of the lungs of the immunized mice were detected to evaluate the protective immunity. The changes results of lung showed that the bacterial counts of DNA vaccine immunized mice were lower than control, especially the pcDNA3.1-ip/pilE group show significantly lower counts than other DNA vaccine immunized groups. The pathologic changes and cell recruitment into the lung of each animal was assessed by light microscopy. In five independent experiments, mice immunized with pcDNA3.1-pilE or pcDNA3.1-ip or pcDNA3.1-ip/pilE decreased significantly infiltration of inflammatory cells in alveolar and interstitial spaces than controls to lethal challenged.This study showed that the three DNA vaccines have higher immugenecity and protective immunity in some degree. It is meaningful to construct fusion gene of candidate genes. This study provided experimental proofs to further study DNA vaccine against Legionella pneumophila.