The targeting study of human serum albumin gene / 生物工程学报

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

The synthesis and function analysis of omega-3 fatty acid desaturase gene from Caenorhabditis briggssae / 生物工程学报

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.

The construction and characterization of human pro-urokinase mutant / 生物工程学报

It is very easy for the pro-UK to lose it's biological activity because of the digestion of pro-UK by the thrombin or the inhibition of pro-UK by the PAI-I. So three pro-UK mutant (pro-UK) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg156 in pro-UK was mutated into His156, and named as pro-UKM1; PAI binding sites Arg178, Arg179, Arg181 were mutated into Lys178, Lys179, His181, named as pro-UKM2; The mutant containing His156, Lys178, Lys179, His181 as pro-UKM3. Three mutants were expressed in CHO cells respectively and analyzed with SDS-PAGE fibrin plate assay and western blot. The results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK2 could resist against PAI inhibition; while pro-UK3 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK3 can be a new medicine of dissolving thrombus.

Exact logistic regression and its performance to SAS system / 中华流行病学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of exact logistic regression, used as a complemental method for the maximum liklihood estimation, and to analyse with data small sample, unbalanced structure and highly stratal nature under the situations of questionable results or inexistence of the maximum likelihood estimation.</p><p><b>METHODS</b>Data from 37 postoperative breast cancer cases were analyzed in 1997 by exact logistic regression under SAS system.</p><p><b>RESULTS</b>Data calculated by SAS software showed that Quasi-complete separation of data points was detected but the results of maximum likelihood estimation did not exist, SAS outputs conflicted the results of the last maximum likelihood iteration (likelihood Chi-square and score Chi-square have similar P, less than 0.05, but the Wald chi-square had a larger P, more than 0.05). Under conditional exact parameter estimation, it appeared that: (1) the joint effect of conditional score statistics was 21.12 with P = 0.000 3; (2) for individual parameters, the effect conditional score statistics of histological classification (grades) was 5.80 with P = 0.020 8; axillary node metastasis (diversion) was 5.74 with P = 0.019 5; tumor size (size) was 0.79, with P = 0.647 6. The effects of tumor histological classification and axillary node metastasis were statistically significant on breast cancer tumour.</p><p><b>CONCLUSION</b>Exact logistic regression seemed to be a very useful method in analyzing data from small sample when the maximum likelihood estimation was either with no effect or did not exist.</p>

Synthesis and function analysis of a new thrombopoietin (TPO) mimic peptide / 中国实验血液学杂志

In order to find a new TPO-mimic peptide with similar activity to TPO while reducing the side effects, a TPO-mimic peptide (P1) screened from a random peptide library was restructured. The new structure of the TPO mimic peptide (P2) was synthesized. After coupling P2 with Dextran 10 and performing intermolecular oxidation, dextran-coupled and dimerized form of P2 were obtained, naming D-P2 and (P2)(2) respectively. The activities of the peptides in vitro were measured with MTT method. The results showed that the EC50 of P2 was 20 nmol/L, 700 times higher than P1. The EC50 of D-P2 and (P2)(2) were 0.35 nmol/L and 0.14 nmol/L, respectively. After administrating to the mouse, the peptides increased the number of platelets in the blood circulation obviously without influence on other blood cells. In conclusion, the TPO-mimic peptides have prospects in treating diseases related with thrombocytopenia.

Screening of TNF-alpha antagonist peptides from a random peptide library displayed with Escherichia coli flagellar / 生物工程学报

Tumor necrosis factor(TNF-alpha) plays an improtant role in the process of anti-infection and anti-cancer. It can both protect and make damage to the host. In order to find new way of inhibiting its host-damaging activity, An E. coli flagella displayed random peptide library was constructed and TNF-alpha antagonist peptides were screened using the peptide library. After 5 rounds of panning and DNA sequencing, six peptide sequences were obtained. Two of them(TBP2, TBP3) have the same sequence frame of V--N-WG. After primary comparation of TNF-alpha binding ability, four peptides were synthesised and purified with RP-HPLC. The activity of inhibiting TNF-alpha was detected with L929 cell and MTT method. The data show that TBP2 and TBP3 can inhibit 90% of TNF-alpha activity when TNF-alpha gives about 30% cell toxicity on L929. The two sequences have not been reported.