Construction Of Prokaryotic Expression Plasmid PET28A/SP-C、PET28a/SP-Aand Expression In Vitro And Purification

BackgroundPulmonary surfactant is a highly surface active complexes composed by phospholipids and proteins. This proteolipidic material is synthesized by type Ⅱ pneumocytes, one of the cell types forming the alveolar epithelium, and follows a regulated exocytic pathway leading to secretion into the thin aqueous layer covering the alveoli.In general terms.mammalian surfactants are composed of85%-90%of phospholipid and8%-10%.of proteins.The protein moiety of surfactant comprises four specific surfactant-associated proteins.The small molecule hydrophobic pulmonary surfactant protein B and C, with the molecular weight respectively6-8KD and3-5KD,and the macromolecular hydrophilic lung surfactant proteins A and D with the molecular weight respectively28-36KD and43KD.The human pulmonary surfactant protein C gene is2.4kb, which located on the short arm of chromosome8with5introns and6exons, only expresses in alveolar type Ⅱ epithelial cells. SP-C gene may originally encode the SP-C protein precursor with191～197amino acids and the protein primary product is21KD molecular weight. After multi-step complex enzymatic, unmature SP-C precursor protein differentiates into the mature and biologically active small molecule hydrophobic protein SP-C with the promotion of the phospholipids in the alveolar surface to form a stable monolayer,playing critical roles in fonnation and stabilization of pulmonary surfactant films, such as reducing surface tension at the alveolar air-liquid interface of lungs in order to avoid alveolar collapse at the end of expiration and to facilitate the work of breathing., promoting pulmonary interstitial liquid reflux。The human SP-A located on chromosome10q21～q24with a nucleotide length of about4.5Kb, consists of two similar genes,SP-A1, SP-A2and a pseudo gene. The nucleotide sequences of human SFTPA1and SFTPA2differ little (94%).So do amino acid sequence (96%). it initially encode248-263amino acids. SP-A monomer is a glycoprotein with a relative molecular weight26-38KD, synthesized and secreted by type Ⅱ alveolar epithelial cells and Clara cell. Natural SP-A is composed of6SP-A trimers and form a "bouquet-like" structure. Its roles are followed①participate in the alveolar surfactant film formation and metabolism, together with the pulmonary surfactant protein B, and promote the surfactant lamellar bodies to converse to the tube myeloid structure, and then extend tube myeloid body to the phospholipid monolayer with small molecule hydrophobic pulmonary surfactant protein B and C;②Raise lipids absorbed by alveolar type Ⅱ epithelial cells with receptor-mediated,inhibit phospholipase secretion, stabilize content of the alveolar pulmonary surfactant;③Increase the antioxidant capacity of the alveolar type Ⅱ epithelial cells;④implicated in surfactant homeostasis and pulmonary innate immunity, it has the chemotaxis and regulate phagocytosis for the immune cells (especially mononuclear macrophages, neutrophils);⑤SP-A and D down-regulation of allergic reaction, and resolution of inflammation, restrain T cell hyperplasia, reduce allergic reaction or allergy or in the lung by other toxic substance (such as silicon dust).pulmonary surfactant protein appear in the cytoplasm of pneumocytes at approximately24weeks of gestation and reach a peak in32-34week.SP-A is the most abundant protein in type Ⅱ alveolar epithelial cells, which account50%in SP, and closely relates to lung maturity. SP-A is closely related to diseases,such as neonatal respiratory distress syndrome (NRDS), adult respiratory distress syndrome(ARDS), pneumonia, asthma, alveolar protein deposition disorder, idiopathic pulmonary fibrosis, sarcoidosis, lung adeno-cancer, Systemic sclerosis and so on. SFTPC gene abnormalities may cause changed content and structural and dysfunction, leading to severe interstitial lung disease such as sarcoidosis, diffuse lung disease and some adult family pulmonary fibrosis, and finally respiratory failure.Now many methods of evaluating lung mature use PS as the analysis object. It can be divided into biological chemical method and biological physics method. Biochemistry analysis method include lecithin/scabbard lecithin (L/S) test, phosphor-lipin acid radical glycerin (PG) test, saturated lecithin determination test and so on; biological physics method judge lung maturity by the surfactant activity of the pulmonary surfactants composition in the amniotic fluid or sputum, including bubble oscillation experiment, fluorescence polarization evaluation FLM, spectrophotometer, LB count and so on. The biochemistry methods of Lecithin/scabbard lecithin (L/S) and phosphor-lipin acid radical glycerin (PG) testing are complex, need lengthy analytical time, it can be vulnerably affected by the pollution of bloody amniotic fluid. Biological physics method all need specific instruments except Biophysics bubble oscillation experiment and has poor accuracy when amniotic fluid was polluted by the blood or vaginal secretion.To be clinically useful, tests for fetal lung maturity should have a high diagnostic sensitivity for immaturity and a high negative (mature) predictive value. So establish a very simple and accurate method which can be widely accepted and used for detecting lung maturity is still a hot exploration.Determining the SP-A concentration is useful for diagnosis and prognosis lung diseases. Du Feng Lan study the correlation of the SP-A’s level between umbilical cord blood and amniotic fluid, demonstrate that SP-A level in the amniotic fluid can be replaced by umbilical cord blood as the sign of infant lung maturity. They measured relative grey value in the NC membrane of different points and seek the actual content of SP-A by curve of standard protein, which is still not able to get precise results. Yoshio Kuroki detecting SP-A in the samples through the method of double antibody sandwich ELISA, detection range from10to1280ng. Since then, the technology of sandwich ELISA is widely used in SP-A test based on SP-A monoclonal antibody. Koji Kaneko had determined SP-A in neonatal peripheral blood、cord blood and amniotic fluid within24h for predicting the neonatal respiratory syndrome, with conclusion that SP-A is an important sign of lung maturity in fetus. For pulmonary surfactant protein C, the main test methods at present is through the gene detection to find mutations so as to confirm diagnosis. Most SP-C protein expression is unnormal in patients with SFTPC gene mutations, while this method is time consuming and expensive.In current domestic market, pulmonary surfactant protein kits and SP-A/C monoclonal antibodies are repacked from abroad. SP-A and SP-C protein are the key step for preparation of kits, the laboratory has obtained high purity and active SP-A from human lung bronchoaleolar lavage fluid, by the method of maltosyl-agarose column chromatography. The content of SP-A extracted from two adult corpse lung is about40mg. But steps are complex ed and the source of corpse lung is difficult.which limited the source of the natural SP.SP-A extracted from lung are used to immune BABL/C to get human SP-A monoclonal antibodies in our laboratory,and product has good immunogenicity. While the source of standard substance for ELISA kits is still difficult.So this research focuses on expression of recombinant protein SP-A and SP-C.by Therefore, a series of experiment studies were performed as follows:obtain target gene by RT-PCR, T-A Clone, The construction of Prokaryotic Vector for PET-28a/SP-A, PET-28a/SP-C, Prokaryotic expression, purification and detection of SP-C and SP-A.Objective:To obtain target gene SFTPA and SFTPC by retrovirus-polymerase chain reaction.Methods:Take the total RNA which is extracted from lung tissue for retrovirus-polymerase chain reaction and detect.with2%agarose gel.Results:Extracting the total RNA from lung tissue successfully, two strip of28s and18s can be seen clearly by agarose gel electrophoresis, the strip of5s is weak, there is no slick of large molecules such as DNA, A260/A280is1.923by Uv spectrophotometer, the2%agarose gel electrophoresis outcome of the obtained purpose gene is consistant with its molecular weight. Conclusion:Obtained target gene of SFTPA, SFTPC successfully by retrovirus-polymerase chain reaction.Purpose:clone PCR product, improve the connection and clone efficiency, of PCR products。Methods:connect the purified target DNA and PMD18T vector, and transform into competence DH5cells, train and form single colonies in the L-AGAR plate medium which contained X-Gal, IPTG, Amp.Count white and blue bacterial colonies.Results:Screening by the Amp resistance and blue white show color, blue white ratio is about16:1, the total number of colonies is about32. Take white single colonies for extraction plasmid after vaccination and amplification. PMD-18T/SP-C plasmid was identified via restriction enzymes and was finally confirmed by the sequencing of the nucleotides.Conclusion:TA clone can clone PCR products successfully.Objective:To establish plasmid of PET-28a/SP-C, PET-28a/SP-A.Methods:The recombinant plasmid PMD-18T/SP-C and PMD-18T/SP-A were cut by restriction enzymes Bam H Ⅰ/HindⅢ and then purified to become SP-C cDNA and SP-A cDNA with viscous ends, as was PET28a. The SP-C cDNA was combined with the PET-28a that had been cut by the enzymes to construct the recombinant plasmid PET-28a/SP-C, The correct PET-28a/SP-C was transformed into BL21to induce expression. Train2h in the800μL LB liquid medium without antibiotic, take50μL coated board (including Kana), train at37℃for one night. Take single colonies by sterile toothpick and train6h in5ml LB liquid medium with50μg/1Amp. Extract Plasmid and cut with enzyme to identify positive restructuring plasmid,finally confirmed by the sequencing of the nucleotides.Results:Recombinant plasmid are sending to gene company for sequencing, it is consistent with the sequence of human pulmonary surfactant related protein gene which published in the gene bank,recombinant plasmid named PET-28a/SP-C, PET-28a/SP-A.Objective:To synthesize、purify and verify SP-C and SP-A protein products.Methods:Plasmid PET28a and PET28a-SP-the A/C respectively transformed into the Ecoli M15[pREP4],training at37℃for the night. Next day transfer it into LB medium with5%, training at37℃for2h, then take3ml bacterium fluid, measure uptake value A600; Join IPTG induction for1～5h, collect bacteria respectively.15%polyacrylamide gel is used for electrophoresis analysis for these express products. With the method of Ni-NTA Slurry (agrose) to purify, take the purified protein collections for western blot and ELISA test.Results:SP-C, SP-A recombinant protein are both single print, no degradation strip. ELISA test show that positive serum OD values of SP-A and SP-C immune animal is greater than2.1times of negative serum, which have good immune response.Conclusion:PET-28a/SP-C, PET-28a/SP-A may be expressed in BL21, and the target protein has no degradation in the process of expression. Successfully constructed Prokaryotic Expression Vector of pulmonary surfactant related protein C/A,and express purpose protein of SP-C/SP-A.