Sequence Analysis Of ITS Region Of Leishmania Donovani Isolates From Different Epidemic Foci In China, Cloning And Expression Of PAL And HSP60 Gene Of Legionella Pneumophila

Leishmaniasis is one of the six key controlled tropical diseases anounced by World Health Organization(WHO). Leishmania donovani (L.d.) is one of the Leishmania complex and cause different types of visceral leishmaniasis (kala-azar ) in the world. According to geographic distribution and source of infection, the leishmaniasis of China may be divided into plain type, hill type and desert type. Different types of kala-azar display differences in the age, effect of treatment, relapse and reservoir host, but the morphology and life cycle of the pathogens are very similar and can not be differentiated, therefore, molecular techniques are required for identification. The ability to distinguish among species is fundamental to the correct diagnosis and treatment. A number of methods have been developed and applied to the question of molecular diversity and relationship within Leishmania, such as kDNA PCR-SSCP, RAPD, analysis of SSU rDNA variable regions, and so on. Ribosomal RNA (rRNA) genes are some of the best target genes.The eukaryotic rRNA are found as tandem repeat units separated by anon-transcribed spacer (NTS) region. The rRNA genes are highly conserved and have been proven value in phylogenetic studies of distantly related species. Among the units, the transcribed, noncoding region of rRNA genes(internal transcribed spacer, ITS) shows extensive variability. ITS evolves much more rapidly than the region encoding the mature rRNAs, have been widely used in comparison of more closely species, even the strains. The ITS region are approximately 1 kb in Leishmania and flanked by highly conserved segments to which PCR primers can be designed.In this study, we used PCR amplification to obtain the two ITS (ITS1 and ITS2) regions located between the small and large subunit rRNA genes (separated by the small 5.8s rRNA ). In order to determine the nucleotide sequence of the ITS region of Leishmania Donovani isolates from desert foci {Ld. XJ771), hill foci (L.d. SCIO) and plain foci (L.d. SD2), and to find out the differences of the sequences of ITS among the three isolates, specific ITS fragments from nuclear DNA of three Leishmania isolates were amplified by PCR and then cloned into PMD18-T vector, finally, sequenced by the dideoxy chain termination method. Sequence analysis showed that the amplified DNA fragments of the three isolates were 1086 bp (Ld. XJ771), 1027bp (L.d. SCIO) and 1028 bp (L.d. SD2). In detail, ITS1 fragments of the three isolates were 230 bp (L.d XJ771), 242bp (L.d SCIO) and 242 bp (L.d. SD2); ITS2 fragments of the three isolates were 603 bp (L.d. XJ771), 531bp (L.d. SCIO) and 532 bp (L.d. SD2); 5.8s rRNA gene of the three isolates were 170 bp (L.d XJ771), 170bp (Ld SC10) and 169 bp (L.d. SD2). 5.8s rRNA gene showed highly conservation (Identities>99%)of the isolates from plain foci, hill foci and desert foci. There were obvious sequence differences among ITS of L.d. XJ771, L.d. SC10 and L.d. SD2. There was a 78 bp special segment of the isolate Ld.XJ771 from desert foci, located between 459~536 bp of the ITS2 fragment. And the isolates L.d. SC10 and L.d. SD2 from hill foci and plain foci were lack of such 78 bp segment. The differences between L.d. XJ771 (desert foci isolate) and L.d. SC10(hill foci isolate) were less than the differences between L.d. XJ771 (desert foci isolate ) and L.d. SD2. (plain foci isolate ). It is the first report of the ITS sequence of the isolates L.d. XJ77J, L.d. SCJO and L.d. SD2 from desert foci, hill foci and plain foci in China. The evidence provided by this study is in accordance with our previous hypothesis about the heterogeneity of isolates from different foci. To some extent, it explains the genetic reason of different appearance of different type of leishmaniasis. The sequence differences of ITS are useful for identification and taxonomy of Leishmania parasites.Legionnaires' disease is an acute bacterial infectious disease which is characterized by severe pneumonia and also an important component of atypical pneumonia in humans .Legionella, the pathogen of Legionnaires' disease, is a facultative intracellular parasite and ubiquitous in natural and man-made aquatic environments or parasites in protozoa. With the popularity of air-condition and humidifier in modern life, Legionella infection will increase and be more severe. The majority of Legionnaires'disease cases are caused by L.pneumophila. Leginella is a common cause of nosocomial pneumonia. Legionnaires'1 disease is acquired by inhaling aerosol contaminated by legionellae. And this disease manifests in two forms: atypical pneumonia and Pontiac fever. The elders, infants and immunodeficient people are susceptible. The bacteria invade alveolar macrophages.where they inhibit phagosome-lysosome fusion and multiply. It was reported in many countries and areas in the world and has been classified as legal communicable disease in many countries, including China. The effective measures of prevention have not been found yet. So it is important for us to do the research of effective and safe vaccine for Legionnaires'disease. Cell-mediated immunity plays a central role in host defense against L.pneumophila, whereas humoral immunity does not appear to play a significant role. Peptidoglycan-associated lipoprotein(PAL) and Heat shock protein 60 (HSP60) are important immunoprotective antigens for L.pneumophila. The aims of our research are to study the cloning and expression of PAL antigen gene, theexpression of Hsp60 antigen gene, and the expression of mixed PAL and Hsp60 antigen gene.In our study, PAL gene of Lpneumophila was amplified by PCR from L.pneumophila serogroup 1, then it was cloned into the eukaryotic expression vector pcDNA3.1(+) and the eukaryotic expression recombinant plasmid pcDNA3.1-PAL was constructed successfully. Sequence analysis showed that the fragment was 531 bp in length and the deduced amino acid sequence is 176. Comparison of the nucleotide sequence with that of Legionella pneumophila in Gene Bank indicated that the identities were above 98%., the identities and positives of the deduced amino acid sequence were above 95% with Legionella pneumophila from the Gene Bank. This recombinant plasmid pcDNA3.1-PAL and another recombinant plasmid pcDNA3.1-HSP60 were transfected into the eukaryotic cell NIH3T3 respectively. At the same time, mixed pcDNA3.1-PAL and pcDNA3.1-HSP60 were also transfected into NIH3T3. Immunofluorescent staining and RT-PCR detection showed the recombinant plasmid pcDNA3.1-PAL and pcDNA3.1-HSP60 could express PAL protein and HSP60 protein in the eukaryotic cell NIH3T3 respectively, mixed pcDNA3.1-PAL and pcDNA3.1-HSP60 could express PAL protein and HSP60 protein in the eukaryotic cell NIH3T3 at the same time. These evidences could be the basis of developing new genetic vaccine for Legionnaires'disease.