| Hypertension is a serious threat to human’s health and becomes the first cause of death. Antihypertensive peptide derived from food is a kind of inhibitor of angiotensin I-converting enzyme (ACE), and decreases the blood pressure by inhibiting the activity of ACE (a key enzyme of blood pressure regulation system). These ACE inhibitor peptides (ACEIP) have been widely concerned, because of their characteristics of moderate effect and high safety. The traditional process of separating ACEIP from natural protein hydrolysates is complex and low-output, but luckily, genetic engineering strategy can overcome these disadvantages and has a promising development prospect. The aim of this study is to use engineering bacterium fermentation technology to prepare the ACEIP Tuna AI (PTHIKWGD) with high activity and stability, through the platform of bioinformatics and genetic engineering. The main content is:to develop computer software based on bioinformatics platform for designing peptide tandem gene and analyzing host environment, to investigate low-cost purification methods of recombinant proteins and peptides, to assess the safety and activity of peptide product and establish a fast detection method for the ACEIP. The main results were as follows:(1)A computer software "Peptide Expression Master" was compiled for small peptide expression. According to the calculated results of the software (using E. coli BL21 (DE3) as the host), the tandem gene unit of Tuna AI was designed and formic acid hydrolysis system was chose for subsequent peptide release. A 4-copy of the Tuna AI gene unit was constructed according to the design scheme. Starting with this gene, the clone vectors pUC-4nTunaAI, the expression vectors pET30-4nTunaAI and engineering bacteria BL21-pET-4nTunaAI (n=1,2,4 and 8) were constructed. After induction, all the genes of Tuna AI were expressed successfully, and the expression level increased significantly with the increase of polymeric level. BL21-pET-32TunaAI showed the highest level, approximately 45.2% of total cell proteins.(2) The method of ultrasonic assisted urea-washing suited for the inclusion body of Fusion-32 TunaAI purification was studied. Compared with Ni2+ affinity chromatography offered by expression vector, the method of urea wash showed better effect in purification (recovery rate increased by 32.6%). At the same time, the study found that inclusion body yield of 33℃,6 h induction condition was only 58.2% of that under 37℃,6 h condition (460.8 mg/L). So the yield of inclusion body that induced at 37℃,6 h and purified by ultrasonic assisted urea-washing method was taken as an index for the optimization of culture medium and culture conditions.(3) The effects of culture medium on yield of inclusion body were studied. Considering comprehensively, LB medium was chosen as the optimum medium among the five kinds of medium (LB, TB, SOC, SOB and 2×YT).10 mM Ca2+, Mg2+, K+ and EDTA were added in LB, respectively. The results showed that only Mg2+ can promote the expression of inclusion body. After IPTG induction,8 high content amino acids of recombinant protein (P, T, G, H, I, K, W and D) was added in medium, respectively, and the result showed the cell density of all groups rose, but only adding the amino acids with high leanness coefficient (H and Y) could significantly improve the yield of inclusion body. After IPTG induction, the highest yield of inclusion body achieved after treatment with sweep frequency ultrasound at frequency of 24±2 kHz for 1.5 min. Finally, optimal culture medium was LB medium containing 10 mM Mg2+, and optimal induction condition was addition of 1 mM of IPTG and 10 mM of tryptophan (Y) and treatment with sweep frequency ultrasound (power intensity of 0.095 W/cm3, frequency of 24±2 kHz, for 1.5 min), the yield of inclusion body can be up to 766.51±44.24 mg/L medium at induction temperature of 37℃ for 6 h.(4)The low-cost purification protocol and safety issue of Tuna AI were studied. After released from recombinant fusion protein by formic acid cleavage, peptide monomer was purified by dialysis, ion exchange chromatography and gel filtration chromatography in sequence, and then identified to be Tuna AI by the HPLC-MS and amino acid sequencing, with a final yield of 351.7 mg/L fermented medium (purity 97%). The results indicated that the obtained peptide Tuna AI had good thermal and acid-alkali stability. To detect endotoxin issue of peptide, ICR mice were given with Tuna AI of maximum concentration (0.4 g/kg body weight) through gavage, no inflammatory response due to endotoxin was detected in experimental animals during 12 h. Given Tuna AI to the ICR mice by gavage 3 times with the some dose, and observed 7 days, the results showed Tuna AI was of safety and had non-obvious side effects (LD50>1.2 g/kg).(5) The antihypertensive effects and mechanism in vivo of recombinant Tuna AI were studied. After single oral administration, the SHR rat systolic blood pressure (SBP) of all Tuna AI group decreased significantly with a dose-effect relationship, but the SBP of normal rats (WKyR) was not affected. The maximal SBP decrease of SHR was observed in high dose group of Tuna AI (2.0 mg/kg body weight), reaching 36.5 mmHg at 6 h, and the antihypertensive effect can be maintained for 10 h and was stronger than captoprilat at 8 and 10 h. The ACE activity of SHR tissues at 6 h post-administration was studied. The results showed that Tuna AI mainly inhibited the ACE activity of artery (inhibition rate was 44.1%), and the ACE activity inhibition of heart, lung and serum was not obviously. Meanwhile, mRNA level of SERCA 2a gene in SHRs heart tissue increased 1.2 times and mRNA of endothelin gene in artery decreased by 31.6%, which indicated that ACE activity was inhibited by Tuna AI after drug administration, reducing the level of Ang Ⅱ that further regulating the expression of gene associated with blood pressure regulation, such as SERCA 2 a and endothelin, to make blood pressure drop. After 35 days gavage, the SBP of SHR was significantly decreased, and the decreased blood pressure reached a steady level after 2 weeks, with the maximal SBP decrease of 41.6 mmHg. Similarly, Tuna AI had no significant effect on the SBP of WKyR. After continuous gavage, no change was observed in the weight gain, whole blood indicators and part serum indicators (cholesterol, glucose, triglyceride) of SHR and WKyR, but the activity of myocardial enzyme of SHR fell by 24.8% and 43.0%, respectively, such as creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase isozymes (LDH1). These results showed that controlling blood pressure by continuous administration of Tuna AI, could reduce some damage of cardiovascular due to high blood pressure.(6) A fast ELISA detection method of Tuna AI was established. The hapten Tuna AI coupled to bovine serum albumin was used as immunogen to prepare antiserum.The high purity polyclonal antibody was obtained from antiserum by two kinds of affinity purification, and applied to establish indirect competitive ELISA standard competition curve. The IC50, detection limit and linear range of this ELISA method was 26.34,0.83 and 1.5 - 364.5 ng/mL, respectively. The range of added recovery and variation coefficient of the Tuna AI in milk, soy milk and tryptone was 82.13%-112.97%,1.76%-7.68%, respectively. All results indicated that the ELISA method can accurately determine the content of Tuna AI effectively. |
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