The Study Of Gene Expression And Molecular Cloning Within ISC10 Gene Of Cryptococcus Neoformans

Cryptococcus neoformans is an important condition for pathogenic fungi, mainly in Yangniao field or bird droppings contaminated soil, in AIDS and HIV-related disease patients cryptococcal the house dust in the air and also C. neoformans can find the pathogens [1]. It often infected with immunodeficiency or immunocompromised patients, thus leading to clinical symptoms, even life-threatening. C. neoformans is different from other human pathogenic fungi, it is a polysaccharide capsule of the dynamic structure of the package, and is able to survive through the immune system of mammals, in the budding reproductive experience when their genes The number, size and sequence of restructuring changes. DNA from the C. neoformans on the level of the etiology and pathogenesis of, for cryptococcal the diagnosis, treatment and prevention provide a more scientific basis. Over the past 30 years, different laboratories to establish a number of eukaryotic molecular biology model system, such as brewer's yeast (Saccaromyces cerevisiae), merozoite yeast (Schizosaccharomyces pombe), the nest structure Aspergillus (Aspergillus nidulans), and so on. These model system to promote the establishment of the C. neoformans the development of molecular biology. The current consensus on the hypothesis that cryptococcal disease, the host may be inhaled through the propagation of being infected, if inhaled a small capsule of bad yeast or sexual reproduction in the natural environment in the 1-2μm the burden of spores . Tam spores is cryptococcal infection propagation of the disease [2], but because of its replication in vitro and animal model of infection has not yet mature. Despite the obvious phenotypic characteristics of the C. neoformans toxic factor has been the identification of related gene has been cloned, but there are still a number of possible toxic factor and its role unknown. C. neoformans ISC10 gene is a new cryptococcal gene products.ISC10 gene (Meiosis-specific protein required for spore formation) is a spore formation of the necessary proteins. 1993, Kobayashi and other [3] isolated from yeast and identified ISC2, ISC10 two-specific gene, discovered ISC2 and ISC10 role in the event of meiosis, have a strong ability to breed sprouting, and confirmed that the gene ISC10 Knockout yeast could lead to a decline in the ability of spores. ISC10 gene from the yeast is separated from the specimen, in artificial induced 7.5 hours after the meiosis, in the synthesis of reproductive cells play an important role. ISC10 synthetic genes can be stable phosphate cellulose, and have contributed to maintenance, rehabilitation and maintenance of the epithelial cells. Research shows that macrophages to the impact of C. neoformans, will lead to the C. neoformans in a certain gene expression changes in the laboratory using yeast gene chip technology to filter C. neoformans has been swallowed up by macrophages before and after the specific expression of the gene , ADK1, CYR1 increase in gene expression, and HSP70, ISC10, such as gene expression dropped [4]. ISC10 gene expression dropped and the C. neoformans was swallowed up by macrophages, the C. neoformans sprouting rate of decline. ISC10 for further in-depth study of C. neoformans and the relationship between the need to be cloned into the C. neoformans ISC10 gene.If directly from the eukaryotic genome DNA obtained gene cloning to express purpose of trying to get the idea will not work, because access to the DNA inside will contain non-coding region. To express eukaryotic expression of the gene and corresponding protein, extracted only through its mRNA and RT-PCR Pofeizhouzhe this way. RT-PCR (reverse transcription-polymerase chain reaction) principle: a total RNA or poly (A) + selective RNA as a template with the cDNA synthesis PCR combination. ART response to the use of reverse transcriptase, a random primers, oligo (dT) or gene-specific primers (GSP) start. RT-PCR method can be one-step or two steps in the form. In the two-step RT-PCR, every step in the best conditions. cDNA synthesis in the first reverse transcriptase buffer, and then remove 1 / 10 of the reaction products for PCR. In the one-step RT-PCR, RT-PCR and at the same time as reverse transcriptase PCR and optimization of conditions, in turn, in a just conducted.This issue through RT-PCR technology from cloned C. neoformans and budding yeast reproduction-related genes ISC10 the full-length cDNA sequence and analysis of biological information, building pGM-T vector recombinant, in E. coli BL21 pLysS in the activity Expression. For the follow-up study of C. neoformans ISC10 gene expression regulation and the potential biological functions laid the foundation for further study of C. neoformans pathogenic mechanism, toxicity and clinical treatment of a new research direction.Research purposes:To prove phenomenon of the mRNA poly glycosides acid (PolyA) tail that total RNA was isolated from the Cryptococcus neoformans strain by reverse transcription-polymerase chain reaction(RT-PCR) . To clone and bioinformatics analyze the full-length cDNA Sequences and nucleotide sequence analysis. of meiosis-specific protein required for spore formation (ISC10) gene from Cryptococcus neoformans , by the reverse transcription-polymerase chain reaction(RT-PCR). To construct recombinant plasmid of pGM-T/ISC10 and transformed into E. coli cells feelings peptide, the study of gene expression activity for the ISC10 gene from Cryptococcus neoformans.Research Methods:To cultivate and isolated and activation of Cryptococcus neoformans wild strains B3501, when the strains cell grow of logarithmic growth phase to collect it, by adding lyase successfully remove the outer wall and capsule of Cryptococcus neoformans. Total RNA was success extracted from Cryptococcus neoformans by yeast RNA Extraction Kit, mRNA of isolation and purification.Using RT-PCR method, reverse transcription synthesis cDNA synthesis template for PCR reaction. To cultivate and isolated and activation of Cryptococcus neoformans ATCC32609 standard strains, the standard strains BLS26, standard strains BLS104, D2Y urea-negative strains (serum type A),wild strains B3501, when the strains cell grow of logarithmic growth phase to collect it, by adding lyase successfully remove the outer wall and capsule of Cryptococcus neoformans. Total RNA was extracted by yeast Kit (RNAsimple Total RNA Kit) method was extracted from Cryptococcus neoformans , according to the first chain cDNA Synthesis Kit (Quantscript RT Kit) method to synthesize. Based on Saccharomyces cerevisiae ISC10 the conservative gene sequence primers were designed and synthesized. PCR amplification of the target gene and PCR reaction products obtained by agarose gel electrophoresis detection, and for the conduct of amino acid sequence and bioinformatics analysis. Cryptococcus neoformans ISC10 coding gene sequences cloned into the expression vector pGM-T vector in the recombinant plasmid, IPTG induction in E. Coli BI21 (DE3) expression, the expression of SDS·PAGE respectively were detected.。Results:Cryptococcus neoformans received total RNA samples tested, the results showed that 28 S rRNA brightness than 18 S rRNA, in the extraction process of degradation has not happened, And in the DNase treatment gel electrophoresis of DNA do not see pollution. Detected by UV spectrophotometer Cryptococcus neoformans D260/D280 to 1.79±0.05, the purity of the RNA better. Through PolyAT tract'mRNA separation system isolated to get the product, using agarose gel electrophoresis and detected by UV spectrophotometry Determination of the concentration, mRNA was not detected presence. To extract total RNA synthesis of cDNA as a template, P1, P2 primers for the PCR conditions, the reaction of the same circumstances, repeated PCR amplification, and the expected results of the same size band, approximately 782 bp fragment encoding 267 amino acid residues, molecular weight of 31.65 KD, in the protein encoded by the 67 amino acid Ala (alanine) mutant Pro (Proline), 233 amino acids from Thr (Thr) for Ser mutation (Ser ). Contain a start codon, two exons of 18 amino acid sequence CO901656, AW782246, CN581440, T17804 is a four-expressed sequence. To clone the full-length cDNA Sequences was meiosis-specific protein required for spore formation (ISC10) gene from Cryptococcus neoformans and to participate in meiosis-specific protein required for spore formation (ISC10) gene from Saccharomyces cerivisiae to compare homology was 99.10%.Cryptococcus neoformans ISC10 gene codon 46 with the LacZ gene (β-galactosidase) with a high degree of integration of similarity. Cryptococcus neoformans ISC10 coding gene sequences in E. coli cells within the expression, the expression vector containing the E. coli pGM-T BI21 (DE3) Cryptococcus neoformans transformants with the cell activity.Conclusion:Cryptococcus neoformans total RNA, DNA contamination without the use of random primer for reverse transcription product of the PCR template for a number of bands.Has been that of total RNA was reverse transcription (using random primers reverse transcription PCR product of the zone also contains anti-retroviral band rRNA). By RT-PCR results demonstrated that the mRNA Cryptococcus neoformans not Poly (A) tail phenomenon, the follow-up to lay the foundation for gene cloning. Using RT-PCR technology, the realization of Cryptococcus neoformans ISC10 gene cloning and identification, and analysis of the Cryptococcus neoformans ISC10 gene sequences. Than on the gene sequence found several highly homologous with the strains and as Detection of yeast in the LacZ gene promoter of the role of a high degree of similarity. This has told us through E. coli LacZ reporter gene to study gene regulation Cryptococcus neoformans and biological functions. Gene expression using recombinant technology, Cryptococcus neoformans ISC10 coding gene sequences cloned into the expression vector pGM-T, T7 promoter downstream Construction pGM-T/ISC10 prokaryotic expression vector. In E. coli BI21 (DE3) was expressed. Cryptococcus neoformans ISC10 gene that these cDNA fragments can be a separate expression, thus verifying ISC10 for a new Cryptococcus neoformans genes for the future lay the foundation for the in-depth study.