To explore the impact of the history of infection by the influenza A virus subtype H1N1 on secondary infection by the influenza A virus subtype H9N2, pigs non-infected and pre-infected with H1N1 were inoculated with H9N2 in parallel to compare nasal shedding and seroconversion patterns. Unlike pigs without a background of H1N1 infection, nasal shedding was not detected in pigs pre-infected with H1N1. Both groups generated antibodies against H9N2. However, levels of H1N1 antibodies in pigs pre-infected with H1N1 increased quickly and dramatically after challenge with H9N2. Cross-reaction was not observed between H1N1 antibodies and H9N2 viruses. These findings suggest that circulation of the H1N1 virus might be a barrier to the introduction and transmission of the avian H9N2 virus, thereby delaying its adaptation in pigs.
Animals ; Antibodies, Viral ; immunology ; Cross Reactions ; Immune Sera ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; physiology ; Influenza A Virus, H9N2 Subtype ; immunology ; Orthomyxoviridae Infections ; blood ; immunology ; Species Specificity ; Swine ; immunology ; virology

Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.

To obtain a recombinant human plasminogen (hPLG) with potential anti-platelet aggregation activity, we cloned the cDNA coding Pro544 to Asn791 of hPLG, a kringle-deficit derivative (hPLG-deltaK). The Pro559 in activation loop was then mutated into Asp559 to provide Arg-Gly-Asp (RGD) motif. The constructed pPICZalphaA-RGD-HPLG-deltaK plasmid was expressed in yeast Pichia pastoris GS115, which produced RGD-hPLG-deltaK about 0.160 g/L broth. After affinity chromatography, the purity of the recombinant protein reached above 90%. Western blotting test confirmed that it retained the immunological reaction capability as human PLG. Its urokinase activation rate in 24 hours and its fibrinolytic activity made no deference against native hPLG-deltaK (P=0.630, n=5). Importantly, after activation by urokinase, RGD-hPLG-deltaK showed a significantly higher platelet aggregation inhibition rate (Ri) (21.8% +/- 1.57%) than hPLG-deltaK (3.8% +/- 0.33%) (P=0.000, n=5). These results proved that we constructed an hPLG mutant with anti-platelet aggregation activity, which made a foundation for developing innovative thrombolytic drugs with multifunction.
Humans ; Kringles ; genetics ; Oligopeptides ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Plasminogen ; biosynthesis ; genetics ; Platelet Aggregation Inhibitors ; isolation & purification ; pharmacology ; Point Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology