Cloning,Identification Of Specificity Genes PCNP And Their Function In The CD8α~+Dendritic Cells

Background：Dendritic cells (DC) are known to be the strongest function in professional antigenpresenting cells and they can activate Non-sensitized T cells. It can be absorbed, processedand passed antigens effectively. Immature DC has strong migration ability and mature DC canactivate T cells of initial type. Mature DC can show a high level of antigen handed a molecule(MHC-I and MHC-II), inducible co-stimulater (CD40CD80and CD86), and itself has highsecretory IL-12. DC usually contribute to the progression and development of a number oftumors, CD8+T cells as the main body of the cellular immune response that the effectiveantitumor immune response, is DC as the basis of immune therapy.Lymphoid tissue of mice CD8α dendritic cells contain three different subtypes at least:CD8+CD4-,CD8-CD4-,CD8-CD4+. The expression of CD8α surface molecular subtypecalled CD8α+dendritic cells (8+DC) and other dendritic cells called CD8α-dendritic cells (8-DC).8+DC exist in the periarterial lymphatic sheath (PALS) and8-DC exists in the edgearea. The two types of DC have common subtype contains CD11c, MHC-I, MHC-II;8-DC’sspecific phenotypes contains CD11b+CD8α-DEC-205-CD4+Mac-1+.8+DC’s surface antigencontains DEC-205, CD8α, CD103.8+DC have several differences of main functionscompared with8-DC:①They can deliver foreign cells antigen and soluble antigen cross thepass to the major histocompatibility complex (MHC-I)；②They are main cells to producedof interleukin-12(IL-12) and stimulate the inflammatory reaction;③When intracellularpathogen infected, they promoted the invasion of pathogens in CD8+T cell response thatbecome the main pathogens antigen;④In the stable state, they are immune regulationperformance and maintain their own organization’s immune tolerance;⑤Specific cell toxicityT lymphocytes (CTL) can induced in8+DC generation that CD8CLT play an important rolein antitumor, HIV/AIDS etc. However we can’t found the type of cells in the human body.French scientists reported in2010, DNGR-1(CLEC9A) and human spleen DC with silmilar inthe phenotype and function compare with mice8+DC. Most important of all, The type of cellscan be multiplied in vitro.In order to looking for differences of8+DC and8–DC, We Screening the two subtypes ofpriority phenotype gene. First of all we get rid of8+DC cells with8-DC the same genes, enrichment8+DC specific genes and establish8+DC cDNA library. From the library the highexpression genes can be cloned in8+DC and low expression or not expression genes in8-DCcan’t be clonbed. We found55kinds of new genes and11species genes not selected genepool of genes, Number14genes (PCNP) significant differentially expressed in the types ofDC.PCNP（PEST-containing nuclear protein）is a kind of newly discovered contains PESTsequence of nucleoprotein. As ubiquitin ligase NIRF (Np95/ICBP90-like RING figerprotein) substrate that NIRF includes a ubiquitin-like domain, a PHD finger, a YDG/SRAdomain and a RING finger. NIRF present high expression in normal cell proliferation and lowexpression in G0/G1in normal cell. As the same time, they persistent high expression in thetumor cells such as HT-1080, HepG2etc.NIRF can be automatic ubiquitination itself. Ubiquitin dependence protein degradation canregulated a variety of cell processes, including cell cycle process, DNA damage, variation andprotein transport. Meanwhile NIRF in9p23-24.1section genes in many types of tumour cellsgenes are frequent. So PCNP and NIRF may with a certain relationship of tumorigenesis.Dendritic cells as an important part of the immune system, it was found in the research hasbeen immunology and even whole life science field the key content. Along with the progressof the research, we found of the distribution of dendritic cells, and the source and function.This paper reviews the mice to lymphoid tissue of CD8α dendritic cells two subtypes ofgenetic differences screening and specificity new genes PCNP screening process. Specificnew genes PCNP function research and translation of the nucleoprotein main part of PESTarea found.Object:The experiment through the gene recombination technology, design the specificity of newgenes PCNP express and silent hairpin structure primer sequence, then two kinds of differentsequence through the different ways to DC2.4transfection, the dendritic cell functionalchanges such as cycle, apoptosis and the influence of other signal path.Methods:Application software technology to design the specificity of new genes PCNP express andsilent hairpin structure primer sequence; Through the RT-PCR detection primer sequencesize, BLAST contrast primer sequences of fit, quantitative PCR detection in the cells PCNPgene expression quantity difference, through the WB technical detection PCNP in cells corresponding amount of protein expression differences; Using flow cytometry test cellapoptosis, growth cycle difference, through the MTT experiment to test the influence of cellproliferation.Results:1, PCNP over-expression and silent primer sequence could be successful designed.2, PCNP over-expression primer success into a eukaryotic expression vector pcDNA3.1(+)3, PCNP silence primer success into a eukaryotic expression vector of pSilencer4.1-CMVneo4, PCNP over-expression and silent plasmid transfected to DC2.45, Using MTT tested cell growth cycle of differences type of DC2.46, Flow cytometry tested cell proliferation cycle differences type of DC2.4Conclusion:We had constructed PCNP expression and silent eukaryotic expression plasmid. Andtransfected into the DC2.4. We found PCNP has important control action on dendritic cells ofgrowth cycle and cell proliferation cycle.