Epidemiology and clinical analysis of 24 children with brucellosis / 中华地方病学杂志

Objective:To analyze the epidemiological and clinical characteristics of children with brucellosis, and to provide a practical basis and experience for clinical diagnosis and treatment of brucellosis.Methods:Retrospective analysis was used to collect clinical data of children with brucellosis diagnosed at the Children's Hospital Affiliated to Zhengzhou University from May 2015 to October 2017, and their epidemiological characteristics, clinical manifestations, laboratory tests, and clinical diagnosis and treatment were summarized.Results:A total of 24 children were included, including 14 males and 10 females, with an average age of 6 years (1 year 2 months to 12 years old). Except February, onset throughout the year, higher incidence was from May to July (14 cases, 58.33%). The exposure history of the children was mainly exposure to cattle and sheep and consumption of beef and mutton (18 cases, 75.00%). The main clinical manifestations were fever in 24 cases (100.00%), bone and joint pain in 14 cases (58.33%), hepatomegaly in 9 cases (37.50%), splenomegaly in 7 cases (29.17%). Tube agglutination test (SAT) was positive in 21 cases (87.50%), weakly positive in 1 case (4.17%) and negative in 2 cases (8.33%). Brucella was detected in all 24 cases by microbial culture, and in 18 cases (75.00%) by blood culture. Eighteen cases (75.00%) had liver dysfunction. Thirteen cases were misdiagnosed, and the misdiagnosis rate was as high as 54.17%. Twenty-two cases had been cured after treatment, 2 cases relapsed and recovered after continued treatment. Conclusions:Children with brucellosis have diverse epidemiology and clinical features, and are easily misdiagnosed. For children with fever, bone and joint pain and exposure history, pediatricians should be alert to the possibility of brucellosis, conduct microbiological and serological tests, in order to timely, accurate and standardized diagnosis and treatment of children with brucellosis.

Expression of human retinol-binding protein 4 in insect baculovirus system and preparation of its polyclonal antibody / 生物工程学报

To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10 000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.

Effects of IL-12 coexpression level on antigen expression and immune responses induced by HBsAg DNA vaccination / 中华微生物学和免疫学杂志

Objective To investigate the effects of IL-12 coexpression level on antigen expression and immune responses induced by HBsAg DNA vaccination. Methods DNA vaccine plasmid pHBV carrying codon-optimized preS2-S gene of reference sequence CHN-HBV07-C in China was constructed. Three DNA vaccine plasmids pHBV-12i, pHBV-12l and pHBV-12h were also constructed by subcloning three different IL-12 expression cassettes with various expression strengths to plasmid pHBV, respectively. Expression levels of IL-12 and HBsAg in vaccine plasmid-tranfected 293T cells were measured by quantitative ELISA. DNA vaccines were administered intramuscularly to BALB/c mice and HBsAg-specific cellular immune responses were determined by IFN-γ ELISPOT. HBsAg-specific antibodies were tested by Chemiluminescence Quantitative Immunoassay. Results The HBsAg expression level in 293T cells was 70 ng/ml when transfected by plasmid pHBV without IL-12 expression cassette, and the HBsAg level was 18 ng/ml when transfected by plasmid pHBV-12l carrying low-level IL-12 expression cassette, whereas the HBsAg level was only 6 ng/ml when transfected by plasmid pHBV-12h carrying high-level IL-12 expression cassette.Results of DNA vaccination revealed that HBsAg-specific humoral and cellular immune responses were significantly decreased in mice administering vaccine pHBV-12h carrying high-level IL-12 expression cassette. Although HBsAg-specific antibody responses in mice inoculated with pHBV-12l were also decreased when compared with those in pHBV-vaccinated mice without IL-12 expression, the HBsAg-specific cellular immune responses were significantly increased. Conclusion High-level coexpression of IL-12 may suppress the expression of HBsAg, Whereas modest coexpression of IL-12 significantly enhanced the HBsAg-specific T cell responses induced by DNA vaccination. Therefore, it is so important to balance the expression between adjuvant and antigen to enhance the immune response.