Prelimenary Study On The Expression Of BPI MRNA And The Cloning Of Bioactive N-terminal Fragment Of BPI

Bactericidal/permeability-increasing protein (BPI) is a type of alkalescence proteins expressed in the neutrophilic granulocytes of mammal. It can bind with the lipopolysaccharide of gram negative bacterium, and increase the permeability of the ectoblast to antibacterial. So it has a biologic effect of counteracting the endotoxin and killing bacterium. It also has a promising prospect on treating the infection of gram negative bacterium.There are two parts of the research. Firstly, nested PCR was used to detect BPI mRNA expression in peripheral blood from 22 patients with CB and 18 normal healthy volunteers. The contents of BPImRNA in the peripheral blood of 22 patients with chronic bronchitis (CB) were semi-quantitatively detected when on admission and after 7 days using Azithromycin Sodium Chloride Injection.. The effect of using Azithromycin Sodium Chloride on the expression of BPImRNA was investigated. And the changing of BPI on the inflammatory process was analysed. Secondly, the gene (BPI₅₉₇) which encode 199 amino acids in the N-terminal fragment of BPI protein were amplified by RT-PCR from the total RNA extracted from polymorphonuclear neutrophils (PMNs) in the human peripheral blood. Then the BPI₅₉₇ gene was cloned into the pBS-T vector, for further study on the role of BPI. Part 1: Study on the expression of BPI mRNAMethods :Nested PCR was used to detect BPI mRNA expression in peripheral blood from 22 patients with CB and 18 normal healthy volunteers. Firstly, the vein blood was collected, and single nucleus cell was separated. Then the total RNA was extracted and reverse-transcripted. The designed primer was used to expand the products of the reverse transcription by PCR. The products of expandition were electrophoresed. Then pictures were taken and analyzed by the VIDAS-21 system. The relative expression amount of BPI was calculated throughβ-actin as an internal standard. The semi-quantitative results were analyzed by Student' s t-test using SPSS10.0. The significant level wasα=0.05.Result:The results of the RNA-extraction were pure and complete. The relative expression levels of BPI mRNA in peripheral blood of 18 normal healthy volunteers were 0.74±0.40. The relative expression levels of BPI mRNA in peripheral blood of 22 patients with CB were 0. 46±0. 23 and 0. 24±0. 19 respectively before and after Azithromycin Sodium Chloride Injection was used. The difference of the relative expression amount between healthy adults and the chronic bronchitis is significant (P=0. 007).Conclusion:Among the healthy volunteers and the patients with CB before and after Azithromycin Sodium Chloride Injection was used, the relative expression amount is different statistically (P<0. 05) . The expression levels of BPI mRNA in peripheral blood of patients with CB were lower after Azithromycin Sodium Chloride Injection was used, indicating that BPI' s antibacterial ability as well as body' s immunological competence decreased after antibiotics were used in inflammatory process.Part 2 : Cloning of bioactive N-terminal fragment of BPIMethods :Total RNA was extracted from whole blood using Trizol. Then it was reverse-transcripted to cDNA. The fragment of the gene which encode N-terminal fragment of BPI protein was amplified by PCR, and the PCR product was cloned into the pBS-T vector. The recombinant plasmid was detected by Ampicillin antibiotic, blue-white screen. The target fragment could be found when recombination was transformed into the competent cells E.coli JM109. The recombinant plasmid was identified. Then the sequence of bioactive N-terminal fragment of BPI was determined and analyzed.Result :The sequence was confirmed by dideoxy-mediated chain in termination. The results showed that the sequence was the N-terminal fragment of the human' s BPI.Conclusion :The construction of clone vector of bioactive fragment of human' s BPI is successful, which is helpful to the further study on the role of BPI.