Effect of rhIL-2 in inhalant form on experimental respiratory tract infection with Klebsiella pneumoniae in mice / 药学学报

<p><b>AIM</b>To determine the protective effect of recombinant human interleukin-2 (rhIL-2) in inhalant form on experimental respiratory tract infection with Klebsiella pneumoniae in mice.</p><p><b>METHODS</b>Mice were infected with the method of nasal intubation drip. During infection, mice were given rhIL-2 by sc injection and the method of nasal intubation drip. There were normal group, vehicle group, model group, rhIL-2 groups and gentamicin group. In the end, the pathological changes in the lung were observed. The survival time and the mortality within a week of each group were recorded. The total protein content, the albumin content, the activity of alkaline phosphatase and the activity of lactic dehydrogenase of broncho-alveolar lavage fluid (BALF) were dertermined and compared.</p><p><b>RESULTS</b>Symptoms of Klebsiella pneumoniae were remarkably relieved because of rhIL-2 administration. The total protein content, the albumin content, the activity of alkaline phosphatase and the activity of lactic dehydrogenase of BALF were less than those in the vehicle group and the model group.</p><p><b>CONCLUSION</b>Inhalation of rhIL-2 can alleviate the pathological changes in the lung after infection. At the same dose, it could be seen that the effect of rhIL-2 in inhalant form was better than that of the injection.</p>

Expression of TMP Fc in Pichia pastoris and identification of its biological activity / 生物工程学报

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZalphaA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/ F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.

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.

Expression of TPO mimetic peptide chimeric proteins with human IgG1 Fc fragments and their biological characters / 生物工程学报

Many antineoplastic agents can cause myelosuppression and thrombocytopenia. Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels. We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo. For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments. The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5' terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5' 648 bp; Fc2: Fc 5' 270 bp; Fc3: Fc 5' 267 bp; Fc4: Fc 5' 90 bp), and cloned into expression plasmid pET28a (+) for constructing four recombinant plasmids. By transforming the four recombinant plasmids into E. coli. BL21 (DE3) respectively, we got 3 kinds of engineered E. coli which express TPO + Fc chimeric proteins(28 kD TPO + Fc1, 12 kD TPO + Fc2 and 12 kD TPO + Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF column. The bioactivities of the expressed chimeric proteins(TPO + Fc1, TPO + Fc2 and TPO + Fc3), TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo, the EC50 on Ba/F3-mp1 cells were 13, 10, 10, 50, and 25 nmol/L respectively, all of them can increase circulating platelet counts. Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO + Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc. These studies have laid basis for production of TPO mimetic peptide by genetic engineering.

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.