| The chief factor contributing to premature neonatal death is respiratory distress syndrome (RDS). Deficiency and dysfunction of surfactant leads to RDS and acute respiratory distress syndrome (ARDS). Replacement therapy with pulmonary surfactant (PS) is the only effective path for the treatment of RDS. Till now, PS was extracted from animal lungs and amniotic fluids, the products of PS can not meet the needs of emergency medicine due to the difficult purification and expensiveness. Synthetic lipids and genetic engineering products of surfactant-associated protein(SP) are the most suitable alternative. Pulmonary surfactant-associated protein A (SP-A) attributes to multiple functions. Human SP-A gene locus locates on the long arm of chromosome 10, q2 1 -q24 and consists of two functional genes, SP-A I and SP-A2, and a pseudogene. PS is a mixture of phospholipids and surfactant-associated proteins SP-A, SP-B, SP-C, and SP-D, which expressed in alveolar type II cells in normal lungs. Secretion and reuptake of surfactant by type II cells are partly regulated by interaction of surfactant protein-A (SP-A) with a specific receptor on type II cells. SP-A is an abundant protein in bronchoalveolar lavage, and it plays most important roles than other SP proteins. Moreover, SP-A is able to regulate the transcription of SPs and augment host anti-microbial defense. Human SP.-A comprises of 248aa and the expressed product of monomeric form is about 26?8kDa. SP-Al gene was subcloned into a prokaryotic vector pGEX-2T and highly expressed in F. coil JM 101 ~JM 105~ JM 109~ XL 1-blue and BL2 1 (DE3). After induced by isopropyl beta-d-thiogalactopyranoside (IPTG), the recombined products, which showed the 52kDa of complete and 4lkDa of 5 incomplete products of GST/SP-A1, accounted for about 6% to 15% of total bacterial proteins on different conditions. At 28 C , only complete GST/SP-A1 proteins was expressed in BL21(DE3). Recombined fusion SP-A1 was about 10% of bacterial proteins. The GST/SP-Al protein was purified according to the purified procedures of inclusion bodies and by Glutathione Sepharose 4B affinity chromatography, which resulted the purity of SP-A1 was about 95%. A 26kDa product of SP-A1 was obtained after digested with thrombin. Anti-SP-A polyclonal antibodies specifically recognized SP-A 1 protein after analyzed by ELISA and Western blot. We concluded that the recombined human SP-A 1 protein was highly expressed in E. ccli and showed its satisfied antigenicity. A yeast recombined vector pVT1O5U/a-SP-A1 was constructed and introduced into Saccharomyces cerevisiae 5-78. This vector contains the yeast a-factor signal sequence, leading to the secretion of expressed protein. 62kDa and 32kDa of recombined products were specifically recognized by anti-SP-A antibodies. The pure proteins were obtained by Sephadex 0-25, Sepharose 4B and HPLC. A methylotrophic yeast Pichia pastoris was also used to produce SP-Al protein. The recombined vector pPIC9/SP-Al was cloned and transformed into P. Pastori. The gene was subsequently integrated into genomic DNA of P pastoris strains OS 115 and KM7 1. Secreted soluble SP-A1 was produced after induced by methanol, which was 32kDa in molecular weight and the products were purified by Sephadex 0-25, Sepharose 4B and HPLC. The SP-A1 protein expressed in Pichia pastoris was efficient in enhancing the phagocytosis of E.coli J5 by alveolar macrophages, wh... |
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