In Vitro Expression Of Secreted HCLCA1and Study In Model Of Human Asthma Airway Epithelial Cell

【Backgroud】The clinical features of asthma are airway hyperresponsiveness and mucushypersecretion, the most important pathogenesis of asthma is chronic, nonspecific airwayinflammation. Mucus hypersecretion is One of the main reasons for the decreasion of lungfunction of patients with asthma and the fatal asthma, but there still have no effectivemeans for its inhabitaion. The pathological basis of increased mucus hypersecretion, is thenumber of goblet cells in airway epithelium. So the study of goblet cells is thebreakthrough point to control the clinical symptoms. Some studies have suggested thatcalcium activated chloride channel I (human calcium-activated chloride channel1,hCLCA1) as a specific protein expressed by new goblet cells, through the regulation of thesynthesis of MUC5AC, have played a irreplaceable role in the synthesis and secretion ofairway mucus.Human hCLCA1and mouse mCLCA3are xenogeneic homologousproteins,in their respective species, they play a key role in airway mucus hypersecretion. Itis considered that they are to be a transmembrane channel protein earlier,, but resenty foreign research group have discovered that mCLCA3and hCLCA1are secreted proteins,and both of them can automatically crack into two independent structures, the75kD ofN-terminal and the35kD of C-terminal.N-terminal as a independent individuals mayplay the function of the full, through regulating the membrane chloride ion channelproteins to achieve the synthesis and secretion of the mucins. In addition, there is a closerelationship between CLCA and airway inflammation, they even may be reciprocalcausation.,so,in vitro expression and the purification of hCLCA1are the basic needand The premise to further research the fuction of it, and it is great help to reveal themechanism of its fuction. that verify the existence form of hCLCA1.【Objective】To observe the expression and secretion of hCLCA1in in vitro epithelial cell model ofNCI-H292cells, explore the possibility of hCLCA1as an airway inflammation medium.Our previous animal experiments have confirmed that the mCLCA3had promotion to theinflammation and mucus secretion in mouse models of asthma. In this paper, we expressedthe N-terminal of a mCLCA3heterogeneous homologous protein, hCLCA1, performedthe protein purification and characterization in E. coli, preparing for the next step ofprotein function research. We also observed the hCLCA1synthesis and automaticcracking in in vitro epithelial cells through infecting NCI-H292cells with hCLCA1over-expressed lentivirus packaging plasmid and did western blotting using thesupernatant and precipitation of cells, preliminarily studied the role of hCLCA1in highsecretion of airway mucus in asthma.【Methods and Results】1. hCLCA1full-length eukaryotic and N-terminal prokaryotic plasmids weresuccessfully constructed.(1)2200bp of N-terminal was inserted into pET28a vector, SalIand Xhol1double enzyme ingestion of recombinant plasmid showed a fragment about2200bp, consistent with expected results.⑵insert full-length of the hCLCA12935bpwas subcloned to pEGFP-N1vector, SalI and BglII double enzyme ingestion ofrecombinant plasmid showed a fragment about3000bp, which was consistent with expected result.The sequencing analysis has showed that the recombinant plasmid EGFP-N1(+)/hCLCA1can be used for the next step experiments.(3)We transfected therecombinant plasmid EGFP-N1(+)/hCLCA1into HEK293cells using Tuberfect, andobserved the green fluorescent protein expression in different time post transfection.Results showed that the hCLCA1was successfully expressed in NCI-H293cells24hourspost transfection. Then cells were collected and nuclear were strained with DAPI toobserve the location of hCLCA1in the cell. Finailly, results has showed that the GFP-hCLCA1expressed in the cytoplasm,nucleus and cell membrane, especially in organellesaround the nucleus.2. The expression, purification and identification of N protein of hCLCA1.(1) pET28a(+)/hCLCA1-N transformation of BL21strain was induced using IPTG, the bestexpression time (5h), temperature (30℃) and IPTG concentration (0.5mmoL/L) were byscreened.(2) Bacteria were induced in the best condition and ultrasonic dissociated on theice, the supernatant and precipitation were respectively detected using SDS-PAGE.Results showed that the pET28a (+)/hCLCA1-N protein was mostly expressed inprecipitation, which accounted for about80%of the total protein.(3) His nickel columnwas used for protein purification, SDS-PAGE electrophoresis showed that high purity ofpurpose protein was obtained. western blotting with His-Tag monoclinal antibody usedshowed a band of about70kD molecular weight,while no band in bacterial proteinsun-induced. Results suggested that the purification of His-hCLCA1-N fusion protein wassuitable for the subsequent experiment, preparing for the next experiment of hCLCA1monoclonal antibodies and subsequent intervention of cell function.3. Lentivirus packaging plasmid of hCLCA1was constructed and infected NCI-H292cells. lentivirus makes up for the poor transfection efficiency of hCLCA1plasmid inNCI-H292cell.(1) to pack helper plasmid and shuttle plasmid packed by lentivirus weretransfected into HEK293cells using Turbofect, immunofluorescence results showedthat GFP expressed well in HEK293cells48h after transfection, and showed highefficiency of infection.(2)The cell supernatant and precipitation were collected48h postlentivirus infecting NCI-H292cells.Western blotting analysis showed that hCLCA1can automatically cracked into N and C parts after synthesis in cells, suggesting that HCLCA1can automatically cracked into two independent structures, N-terminal and C–terminal,and both parts might undertake different functions of hCLCA1. It laid the foundation forthe later research of lymphocyte chemotaxis and adhesion function of secretory protein insupernatant.【Conclosion】1.We synthesized the active peptide of human hCLCA1-N, and performed the prokaryotic expression and purification of the protein, laying a foundation for the preparationof hCLCA1antibody.2. In order to observe the synthesis of hCLCA1in epithelial cells and whether it cancrack intracellular and secret to the extracellular, we packaged the hCLCA1in a viral vector and infected NCI-H292cells, results showed high infection efficiency about higherthan60%.3. The protein location in the cell was observed after infection of NCI-H292cellswith the viral vector, results showed that hCLCA1can be found cracking in the in vitromodel of human airway epithelial cells, and was secreted extracellularly, suggesting thathCLCA1may have certain effect in nonspecific inflammation of asthma respiratory epithelium cells.