An Investigation On Serum Antibody To Legionella Among People In Chengdu And Cloning And Expression Of HSP60 Gene Of Legionella Pneumophila

Legionnaires' disease is an acute bacterial infectious disease which is characterized by severe pneumonia and also an important component of atypical pneumonia in humans . It severely engangers human health. It was reported in many countries and areas in the world and has been classified as legal communicable disease in many countries. Legionella, the caustive agent of Legionnaires' disease, is a facultative intracellular parasite and ubiquitous in natural and man-made aquatic environments or parasites in protozoa. Leginella is a common cause of both community-acquired and nosocomial pneumonia. With the popularity of air-condition and humidifier in modern life, Legionella infection will increase and will be more and more severe. The genus Legionella currently contains 42 species and 64 serogroups. And the majority of Legionnaires' disease cases are still attributed to L. pneumophila. Disease is acquired by inhaling aerosol contaminated by legionellae. The bacteria invade alveolar macrophages, where theyinhibit phagosome-lysosome fusion and multiply. And this disease manifests in two forms: atypical pneumonia and Pontiac fever. If patients are treated incorrectly, the mortality rate is very high. The epidemiological investigation findings indicate that people in our country are infected with different serogroups of legionella. China emphases prevention and cure Legionnaires' disease as a new emerging infectious disease. But any good way to deal with it has not been found. Cell-mediated immunity plays a central role in host defense against L. pneumophila, whereas humoral immunity does not appear to play a significant role. Heat shock proteins are immunoprotective antigens for many microbial pathogens. HSP60 is the most abundant protein synthesized by intracellular L. pneumophila organisms. It expressed on the surface of L. pneumophila. A previous study reported that HSP60 of L. pneumophila can induce cell-mediated immune responses and protective immunity against a lethal challenge with L. pneumophila in the guinea pig model of Legionnaires' disease. The gene coding HSP60 is a promising candidate for the development of a DNA vaccine against legionellosis. The aims of this study are to investigate serum antibody to legionella among people in Chengdu, and to study the cloning and expression of HSP60 antigen gene of legionella pneumonia.1 An investigation on serum antibody to legionella among people in Chengdu393 human serum samples, which were collected from healthy people and patients suffering pulmonary diseases, were detected for antibodies to Legionella using mi croaggluti nation test (MAT) with antigens of Lpl-8,Lm. So we can obtain the data about the status of Legionella infection in Chengdu. The results are summarized as follows: Significant antibodies to Legionella were tested in all 9 serogroups and species. It was 10.82% that one person was infected with two or three serogroup. The positive rate of Legionella antibody in the healthy people was 31.60%(73/231) and 35.80%(58/162) in patients suffering pulmonary diseases. The positive rate of leginella of two groups showed no statistical difference (P>0.05). Lpl in the healthy people was most commomly (17.31%), and next was Lp6 (11.26%). The rate of Lp8 was the lowest (0. 43%). The positive rate of Lpl in patients suffering pulmonary diseases was most commomly (12.35%), and next was Lp6 (5.56%). The infective serogroups were the same in the two groups. It indicated that people in Chengdu were generally infected with different serogroups of Legionella. This study is the first report that to obtain the data about the status of Legionella micdadei infection in Chengdu.2 Cloning and expression of heat shock protein 60(HSP60) gene of legionella pneumophilaHSP60 gene of L. pneumophila was amplified by PCR from a template of L. pneumophila serogroup 1. The amplified DNA was cloned into pUC18 vector and the recombinant plasmid pUC18-HSP60 was constructed. The results of sequencing showed that the fragment was 1647bp in length. Comparison of the nucleotide sequence with that of legionella pneumophila in Gene Bank indicated that the identity was 98%. The deduced amino acid sequence is 548, its identity was 99% with legionella pneumophila from the Gene Bank. 60kD protein of pUC18-HSP60 was inducedto express in prokaryotic cell. The target gene in the recombinant plasmid (pUC18-HSP60) was sub-cloned into eukaryotic expression vector pcDNA3.1(+) and the eukaryotic expression recombinant plasmid pcDNA3.1-HSP60 was constructed. This recombinant plasmid was then transfected into the eukaryotic cell NIH3T3 and the expression of HSP60 gene in eukaryotic cell was detected by immunofluorescent staining. Immunofluorescent staining of recombinant plasmid transfected NIH3T3 showed positive reaction. So we concluded that the recombinant plasmid pcDNA3.1-HSP60 could express HSP60 protein in eukaryotic cell NIH3T3. Until today, this study is not reported domestic. And it laid a good foundation of the research of a new genetic legionellosis vaccine.