|
Peptide antibiotics (Peptide antibiotics) organism gene encoding the small peptide, usually constituting from 12 to 60 amino acid residues, a molecular weight of less than 5 kDa. They are moving, are widely distributed in the plant, suggesting that play an important role in the growth of the organism and the anti-infective. Although discovered the anionic peptide antibiotics, but the vast majority of peptide antibiotics cationic. Defensins such as animal (Defensins) is a group of cationic peptide antibiotics, it is first found from the rabbit neutrophils, and found in macrophages of different animals, the paneth cells of the small intestine, epithelial cells also expressed, having anti-bacterial, anti-viral, anti-fungal and other functions. The mammalian defensins divided into alpha-, beta-, and θ-three subfamilies depending on the manner of distribution and the formation of disulfide bonds of the cysteine. Human defensin molecules contain six cysteine ??residues, can form three disulfide bonds. Defensin 17 kinds of people, including six α-defensins (HNP1-4, HD5 and HD6) and 11 β-defensins (HBD1-6, and HBD25-29) have been found so far. The body being infected, they play an important role in the body of specific immunity before. In clinical resistant increasingly serious problem, no doubt to the clinical anti-infective therapy of virus infection, the lack of specificity of treatment, the discovery and in-depth study of peptide antibiotics lit a new hope. Peptide antibiotics hPAB-β human hBD-2, a mutant, in our previous work by removing the hBD-2N-terminal three amino acids, added at the C-terminal of an asparagine obtained having hBD-2 Similar antibacterial activity. In previous work, we use as of Pseudomonas aeruginosa the phage PaP3 gene coding PaP3.30 protein carrier molecule fusion hPAB-β expression of fusion protein expression rate of 40% of the total bacterial proteins, but the fusion protein molecular weight (23.6 kDa) is a target peptide hPAB-β (4.3 kDa) five times, so the actual hPAB-β expression is less than 7%, the expression efficiency is not high; the later series body (forthcoming hPAB-β gene connected end to end, to build different copy number of the expression vector), but because a hPAB-β contains six cysteine, the series more molecules, the more complex the formation of disulfide bonds, the fusion protein formed inclusion bodies The solubility of the worse, it is difficult to achieve the efficient preparation of hPAB-β; Finally, according to the Pichia pastoris expression of exogenous protein yield and secreted expression, we have successfully built and peptide antibiotics hPAB-β in Pichia pastoris secretory expression in the target polypeptide production reached 241.2 ± 39mg / L, the paper reported the purification of the the yeast high density fermentation peptide antibiotic hPAB-β, and in high purity hPAB-β, herpes simplex virus as a model, role in the killing of hPAB-β herpes virus, the mainly experimental methods and results are as follows: 3.7L fermenter recombinant the peptide antibiotics hPAB-β expression cassette pPIC9K-take advantage of the the Pichia pastoris easy high density fermentation characteristics yeast excellent 5 hPAB-β/GS 115 engineering bacteria for high density fermentation, growth in the cell reaches a certain amount, methanol-induced expression of the target gene and cell growth curve, monitoring the level of expression of the target protein. Be seen from the growth curve, within 16h after seed inoculation, yeast growth rapidly join the feeding yeast to continue to grow, stop feeding the growth of 32h (the bacterial wet weigh up to 236.7 ± 25.3 g of / L) cell growth slowed down during methanol induction, slow cell growth. 8-12h in the induction process, the interval sampling Tricine SDS-PAGE electrophoresis analysis, the results can be seen in the beginning of induction, supernatant no goals peptide secretion addition of inducer 12h, vaguely see the expression bands and then, expression with gradually enhanced to the highest induced 60h under tank. The total protein concentration were measured three times the fermentation broth using the Brandford France 664.0 mg / L, 781.8 mg / L and 721.3mg / L. Band detection using the Bio-Rad's Quantity One software strip the gradation analysis tools after gel electrophoresis of the protein bar band analysis target protein accounted for about 33.4% of the total protein is fermentation supernatant, it was found that the target protein in the fermentation supernatant The expression about 241.2 ± 29.5 mg / L, to achieve the goal of high-density fermentation of recombinant Pichia pastoris expression peptide antibiotics hPAB-β. Purification purposes is not only to obtain the high purity of the target product, but also so that the target product does not lose its biological activity during the purification process, which would be based on the difference in design of the target molecules and impurity purified route tested groping be optimized. Taking into account the hPAB-β as a cation of the small peptides (PI 9.001), we designed a 10kDa membrane filtration, reverse phase chromatography, cation exchange, purification techniques such as molecular sieve desalted purified target peptide, final purification obtained from the per liter of fermentation broth 74.9 ± 1.5 mg target protein purity of more than 98% of the overall purification efficiency of 31.5%, the separation and purification of the yeast expression of peptide antibiotics hPAB-β. Obtain purified hPAB-β by Western blot identification of the target peptide, the target product could hPAB-β-specific antibody response, indicating that the purified protein is the need object. Further use of the agar diffusion method detection overcast purified product against Gram positive bacterial killing activity, results showed that the purified product with biological activity, and laid the foundation for further development of the study of the function of the hPAB-β. Virus isolation and typing the \With the progress of genomics, the herpes simplex virus genome sequences have been clearly laid a good foundation for the application of molecular biological diagnostic techniques (PCR, nucleic acid probes, etc.) in the diagnosis of herpes simplex virus infection. For the study of peptide antibiotics hPAB-β antiviral activity, we have to herpes simplex virus as a model for virus isolation were collected from 60 cases of clinical specimens suspected genital herpes lesion, and synthesized according to the herpes simplex virus DNA polymerase gene sequence type-specific PCR primers, PCR analysis of clinical specimens and compared with the cell culture. Results 60 specimens were suspected in patients with genital herpes, HSV type specific PCR, 45 were seen approximately 390bp specific amplification product (HSV-2), the positive rate of 75%, while HSV-1 specific primers detected as negative; Vero cells for virus isolation and culture positive in 36 cases, the positive rate of 60%, and PCR, the difference was significant (X2 = 24.38, P lt; 0.01). From the type of skin lesions of patients, blisters and pustules specimens, viral culture and PCR positive rate was higher (80-93.3%), erosion, crusting rash specimens tested positive for low rate, especially rash lowest prompted etiological diagnosis of suspected patients with genital herpes, acquisition blisters, pustules specimens for virus isolation or PCR detection better. Using recombinant the Pichia Yeast pPIC9k-hPAB-β/GS115 high density fermentation, purification, and preparation of the peptide antibiotic hPAB-β. Dilution method to detect the recombinant hPAB-β toxicity on Vero cells, the results show a high concentration of peptide the antibiotic hPAB-β (1 mmol / L or more) toxic to cells on Vero cells, the maximum non-toxic concentration of 400μmol / L about 1.72mg/ml. In clinical specimens isolated HSV-2 as an object of study, the peptide antibiotics hPAB-β acts on cells inoculated after HSV-21.5h results visible dilution (400-10μmol / L) of each experiment, peptide antibiotics have inhibit viral macrophage plaque-forming ability, with the peptide antibiotic concentration increased plaque formation to reduce the number, indicating that the high density by yeast fermentation hPAB-β peptide antibiotics against clinical isolates of HSV-2 has a direct role in the killing. On cultured hole After the virus liquid REF (100TCID50 virus fluid 0.2ml PBS 0.2m1) began to appear in culture 20h i.e. cytopathic cultured 2d, with 1% crystal violet (containing 10% formaldehyde) staining, denoted by the average number of plaques formed The number of 101.7 ± 5.5. Under the same experimental conditions, the high concentration holes to acyclovir are reflected in its viral suppression effect, when the concentration was reduced to below 100μmol / L, the virus plaque forming the number did not change significantly. Based on this, the chosen concentration of 200μmol / L peptide antibiotics hPAB-β and acyclovir, antiviral activity of the comparative analysis, the plaques of the virus control group was 100% is formed, and the peptide group of antibiotics hPAB-β effect macrophage plaque formation was 48.7%; 200μmol / L of acyclovir role group plaque formation rate was 39.0%; virus plaque-forming of two drug action group has a significantly lower compared with the virus control group (P = 0.02545 and 0.0103), the the peptide antibiotics role of group compared with the control group of acyclovir drug, plaque formation rate is slightly higher (48.7% of the former and the latter 39.0%), but there was no statistical significance (P = 0.06927) . HSV-2 show that the peptide antibiotic hPAB-β has a good killing activity. In summary, the use of high density fermentation of recombinant Pichia pastoris reached 241.2 ± 29.5 mg / L, the amount of peptide antibiotics hPAB-β expression by 10kDa membrane filtration, reverse phase chromatography, ion exchange, size exclusion chromatography, and other processing the high purity of the target product was purified yeast fermentation supernatant 74.9 ± 1.5 mg / L, the overall efficiency of the purification of 31.5% of the purified product against both gram positive and negative bacteria's ability. Using PCR, virus isolation and culture of the detection and identification of specimens of 60 suspected cases of patients with genital herpes, 45 PCR products of positive, positive rate of 75%, both HSV-2 and HSV-1-specific primers tested negative; Vero cells, virus isolation, the results of 36 cases were positive, the positive rate of 60%, compared with PCR detection, the difference is very significant. Using a virus plaque counting method hPAB-β anti-viral activity of the peptide antibiotic into the analysis, the results under 200μmol / L hPAB-β or acyclovir when the role of the former virus plaque formation was 48.7%, after by 39.0%, compared with the virus control group, both significantly decreased, compared to the peptide antibiotic hPAB-β with acyclovir group, the formation of the virus plaques suppressed without significant differences (P = 0.06927), showed that yeast expression and purification of peptide antibiotics hPAB-β has anti-HSV-2 activity, and provide an experimental basis for in-depth analysis of its antiviral mechanism.
|