Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.

New strategies are needed for prevention of Pseudomonas aeruginosa (P. aeruginosa) infections, a widespread disease caused by P. aeruginosa with strong drug resistance. The immunoprotective capacity of the receptor of autoinducers protein LasR/RhlR was examined in the BALB/c mice. At first, specialized expression plasmids were developed to facilitate expression of LasR/RhlR proteins in Escherichia coli (E. coli). Then, biofilms were grown from clinical isolated P. aeruginosa PA0305 to investigate the relative contributions of cell signaling for biofilm formation. Morphological characters of biofilm were quantified using Image-Pro Plus software. Fluorescence analysis demonstrated that cell signal molecule LasR/RhlR significantly (P ＜ 0.05) influenced development of P. aeruginosa biofilm. Active immunization of mice with LasR/RhlR was found to provide significant protection against homologous challenge with P. aeruginosa in mice lungs. In 10 days after lungs inoculation, the bacterial clearance rate of the immunized mice was clearly higher than that of non-immunized groups on the basis of microbiological and histological assays. The protective effects of immunization with LasR and Rh1R together were the same as the result of LasR or Rh1R immunized mice alone. These data indicate that the manner ofLasR, Rh1R or both is an important determinant of immunoprotection in mice lungs infection.

Mannheimia haemolytica is an important pathogen causing respiratory diseases in sheep,and the development of an inactivated vaccine of sheep origin against Mannheimia haemolytica is an effective means of prevention and control of this pathogen.In this study,Mannheimia haemo-lytica isolated from the diseased material collected from a sheep farm was screened for the most suitable vaccine strain by mouse pathogenicity test,and the inactivated vaccine was prepared by combining four antigenic amounts(106,107,108,109 CFU/mL)with three adjuvants(ISA 201 ad-juvant,ISA 206 adjuvant,and aluminum adjuvant),which were used in immunization and attack protection tests,and then screened for immunity and attack protection tests.Immunization and at-tack protection tests were conducted to screen the optimal antigen amount and adjuvants for inacti-vated vaccine preparation.Finally,the vaccine was prepared under the optimal conditions and the dynamic changes of antibody levels in immunized sheep were observed.The results showed that a total of 24 strains of Mannheimia haemolytica of sheep origin were isolated,among which 12 strains of serotype A1 were the dominant serotypes for the infection in this sheep farm;one strain of Mannheimia haemolytica of sheep origin of type A1(Mh1)was screened and obtained as the strain for vaccine preparation.Its LD50 for mice was 3.16 × 107 CFU/mL,with strong pathogenici-ty,and could cause pathological damage such as inflammatory cell infiltration of varying degrees in heart,liver,spleen,lung and kidney tissues;the results of the immunization and attack protection test showed that the optimal antigenic amount of the inactivated vaccine was 109CFU/mL,and the most suitable adjuvant was ISA 201,which stimulated the production of the highest antibody level and provided 100％protection for the mice.The protection rate of mice reached 100％,and good humoral immunity could be induced after immunizing sheep.The inactivated Mannheimia haemo-lytica type A1 vaccine prepared in this study has good immunoprotection effect,and lays a good foundation for the further development of commercial vaccine against Mannheimia haemolytica of sheep origin.

This is a study on the biodegradable polymers as gene controlled-released coatings for gene transfer. The PELA (poly (Dl-lactic acid)-co-poly (ethylene glycol), and PLGAE (poly (lactic acid)-co-poly (ethylene glycol)-co-poly (glycolic acid) random copolymer) were synthesized and prepared as the coatings of plasmid pCH110 in the transfection. All kinds of factors affecting the loading efficiency, cytotoxicity, transfection efficiency and the course of the degradation and release in vitro were discussed. The average diameters of microspheres of PELA and PLGAE were 1-3 microm and 0.72 microm respectively. The loading efficiency levels of them were 62% and 70% respectively. The transfection efficiency levels of two kinds of pCH110 delivery system for COS-1 cells were higher and two of them had few cytotoxicity. After transfection, the X-gal assay was performed and reported positive for 96 h. The biodegradable polymeric materials as gene carriers possess their potential superiority.
Biocompatible Materials ; DNA ; chemistry ; Drug Carriers ; chemical synthesis ; chemistry ; toxicity ; Gene Transfer Techniques ; Lactates ; chemical synthesis ; chemistry ; toxicity ; Lactic Acid ; chemical synthesis ; chemistry ; toxicity ; Polyethylene Glycols ; chemical synthesis ; chemistry ; toxicity ; Polyglycolic Acid ; chemical synthesis ; chemistry ; toxicity ; Polymers ; chemical synthesis ; chemistry ; toxicity ; Transfection