| Uncontrolled inflammatory response leading to immunity dysfunction and septic shock, even multiple organ dysfunction syndrome, is one of the most critical event in clinical patient' mortality. In recent years, among the research of the inflammatory signal transduction, a new regulatory protein named A20 was recognized. It down-regulate the expression of TLR4, an important membrane receptor in inflammatory signal transduction, and can also down-regulate the activities of nuclear transcription factor NF-κB and AP-1. So, it plays a crucial role in keeping the balance between pro-inflammatory and anti-inflammatory cytokines and was taken as one of the intrinsic protecting proteins for tissues and cells. Regarding on the urgent drug requirement in clinical therapy of sepsis and the clinical diagnosis requirement for detecting the expression levels of A20 in samples at the first moment by ELISA, we designed a rhA20 molecule under the theory and methods of molecular design. We put great efforts to carry out a series of researches around expression A20 in Pichia pastoris from constructing the expression vector (yeast expression vector for cell-penetrating peptide fusion protein, yevCFP) as well as cloning human A20 cDNA. These are essential foundations for studying the anti-inflammatory effects and molecular mechanism, and preparing monoclonal antibody of rhA20. The latter is ready to develop a diagnostic ELISA kit for detecting the levels of A20 in clinical samples. The main research methods, results and conclusions were summarized as follow:A. Based on the molecular designing methods, we selected a regime of rhA20. Bioinformatics analysis suggested that the predict secondary structure,important physical and chemical characters of designed rhA20 relatively approach to natural hA20. The full-length of designed rhA20 is 809 amino acids. The predict molecular weight is 93kD.B. Total RNA of peripheral blood leucocytes stimulated by LPS was isolated and purified. Multi-pair PCR primers were designed. The 2361bp cDNA fragment of hA2O ORF was cloned by nested gradient RT-PCR and inserted into pCRR4-TOPOR vector by genetic engineering method. The results of sequencing analysis confirmed that the sequence of 2362bp cDNA fragment is the same as that in GenBank.C. Using our designed single-primer PCR, after many alternation amplification with different single primers and subsequently degenerating and annealing of their corresponding products in a PCR tubule, we successfully prepared a considerable quantity of CPP-Linker with 80bp length. D. Using genetic engirneering methods such as endonuclease digestion, DNA ligation catalysed by T4 DNA ligase,transformation and colony PCR etc, we successfully constructed yevCFP. The expression plasmid of rhA20, called yevCFP-rhA20, was obtained after insert the 2363bp DNA fragment into the yevCFP vector.E. Electroporation of Pichia pastoris was carried out with linearized yevCFP-rhA20. High copy clones were obtained through screening with MD/-His and YPD/G418. Pichia integrnants with full hA20 ORF gene were confirmed by PCR. The rhA20 was expressed by Pichia pastoris strain GS115 with methanol induction. The molecular weight of expressed rhA20 is about 93kD. The full expressing products hold 1.1% in total proteins of expressing supernatant and the expressing amount achieves to 22.84±0.17μg/ml.F. The recipe of BMMY medium was reformed and the culture condition was optimized. It was found that the degradation phenomena of expressing products can be controlled by a certain extent of decreasing the pH of medium, adding a low dosage of EDTA, adding the substrate of protease, as well as decreasing the induction temperature. G. The purified methods of expressing rhA20, including ultrafiltration and (NH4)2SO4 precipitation, were established. H. Using immunofluorescence staining, the laser confocal microphotographs showed that rhA20 could come into THP1 cells in 40 minutes and the distribution of rhA20 was mostly in cytop...
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