Expression of Ietalurus punetaus β-defensin based on recombinant Pichia pastoris / 生物工程学报

β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.

Comparison of the Improved Method and the Traditional Method for Toluidine Red Unheated Serum Test in Syphilis Screening / 现代检验医学杂志

Objective To compare the detection performance of the modified serum test (TRUST) method and the traditional method in syphilis screening.Methods A series of TRUST high titer syphilis serum was diluted,and the positive rate of each method was calculated by using the improved method and the traditional method.Comparison of two detection methods of C50,C5 ～ C95 interval,as well as the accuracy of the density curve,and the consistency of the two methods were compared,and diagnostic performance were compared.Results The improved method of C50 was less than the traditional method of C50,and the improved method of C5 ～ C95 range was narrower than the traditional method,compared with the traditional method.The improved method of the non precision density curve was steeper than the traditional method,and the two confidence interval of the consistency degree of the 95％ methods was 73.4％ to 95.8％.The diagnostic sensitivity (SEN),diagnostic specificity (SPE),positive predictive value (PPV),negative predictive value (NPV) and diagnostic efficiency(DF) of the improved method were 64.29 ％,99.1％,85.71％,97.05 ％ and 96.39 ％,respectively.The SEN,SPE,PPV,NPV and DF of the traditional methods were 3.75 ％,98.49 ％,75 ％,96.18 ％ and 95 ％,respectively.The improved method was superior to the traditional methods in the two aspects of SEN and PPV (x2 =8.25,10.03,all P＜0.05),with the statistically significant difference.The improved method was slightly higher than the traditional method in SPE,NPV and DF (x2 =2.39,3.45,4.03,all P＞0.05),with the no statistically significant difference.Conclusion The precision,diagnostic sensitivity and diagnostic specificity of the improved method was higher than that of the traditional method,and it can be applied to the detection of large batch samples with the aid of the full automatic enzyme immunoassay instrument.The improved method can be used to replace the traditional method for syphilis screening.

Influence of different contact ways and extracting conditions on the hemolytic effect of biomaterials / 生物医学工程学杂志

This assay was aimed to evaluate the influence of different contact ways and extracting conditions on the hemolytic effect of biomaterials. Using direct contact method and extract contact method, we assessed the hemolytic effect of PDLLA and PVC. The extracting conditions included: 37 degrees C 24 h, 37 degrees C 72 h, 37 degrees C 120 h, 50 degrees C 72 h, and 70 degrees C 24 h. After the material or extract had been in contact with the diluted blood of rabbit for certain times, the hemolysis rate was calculated. The results for PDLLA showed there were some differences between direct contact and extract contact at 37 degrees C for different extraction time (P < 0.05), but the hemolysis rates, lower than 5%, were in accord with the requirements of medical devices. However, under the condition of 50 degrees C and 70 degrees C, there were significant differences when extract contact method was compared with direct method (P < 0.01). For PVC, there was no statistically significant difference under all conditions (P > 0.05). Our conclusions: (1) Under the extracting condition of 37 degrees C from 24 h to 120 h, the soluble part of PDLLA and PVC that might influence erythrocyte did not dissolve considerably. (2) Under the extracting condition of 50 degrees C and 70 degrees C, the hemolysis rate may remarkably vary with the chemical characteristics of tested materials; (3) As to an unknown material, it is advisable to adopt two methods at the same time, one for direct contact and the other for extracontact. Thus the hemolytic effect of biomaterials can be evaluated from physical and chemical angles. (4) In case that the chemical property of the sample can endure the test, the extracting condition at 50 degrees C and 70 degrees C may be of benefit to assessing the hemolytic effect of biomaterials. (5) The extract contact method as a supplemental test of direct contact method is of realistic significance.

Evaluation of the short-term biocompatibility of a new kind of hydrogel prosthetic nucleus / 中国组织工程研究

Aim To evaluate the short-term biocompatibility of a newkind of prosthetic nucleus-Evergel, which is made from the modifiedpolyvinyl alcohol hydrogel. Methods According to China national standardGB/T16886 documents, the toxicity of Evergel prosthetic nucleus materialwas investigated by the cytotoxicity test, sensitization test, haemolysis test,Ames test, mice marrow micronucleus test and chromosome aberration test ofmammalian cell in vitro. Results This material had no cytoxicity, no sen-sitivity, no obvious haemolysis, and no mutagencity in Ames test, micemarrow micronucleus test and chromosome aberration test of mammalian cellin vitro. Conclusion The Evergel prosthetic nucleus has a good biocom-patibility and can be used clinically.