Objective To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.Methods Total RNA was isolated from unfed female ticks,mRNA was purified and a library of oligo(dT)-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA.The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the ?SCREEN vector.Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8.Recombinant plasmids that were subcloned to E.coli BM25.8 were isolated and transformed into E.coli JM109.Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.Rusults The recombinant phage DNA was packaged by using phage-marker packaging extracts,resulting in a primary cDNA library with a size of 1.8?106 pfu.Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4?109 pfu/ml.Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library.Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H.longicornis tropomyosin mRNA,Rhipicephalus annulatus unknown larval protein mRNA,chromosome 2R of Drosophila melanogaster,mitochondrial DNA of H.flava,clones HqL09 unkown mRNA and Hq05 mRNA of H.qinghaiensis,and myosin alkali light chain protein mRNA.Conclusion The cDNA expression library from unfed female H.longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

Objective To establish the real-time quantitative PCR (RQ-PCR) assay for detecting Candida albicans (C.albicans) in whole blood and its clinical application in the febrile surgical patients who may develop gut barrier damage and gut microorganism translocation.Methods The NAG1 gene,which is a single copy in C.albicans genome,was selected as the target gene for designing the primers and probe.The plasmid was fabricated and produced as standard samples.C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the total 20 μl TaqMan RQ-PCR amplification reaction system was established.The 74 venous blood samples from the surgical febrile patients were detected for C.albicans load.Results The specificities of the primers and probe were excellent,the correlation coefficients of the standard curves were between 0.9918 and 0.9985,and the efficiency of amplification was 0.88-1.027 for the samples above the lowest detection limit (100 copies/μl examine fluid,or nearly 1.1 × 103 cfu/ml whole blood).The average accuracy of the RQ-PCR equipment was (99.64±2.08) %,the sensitivity was 97.46%,the specificity was 100%,and the average coefficients of variation (CV) of the intra-and inter-assay were (14.76±2.64)% and (17.85±3.53)%,respectively.The average recovery rate of C.albicans DNA in whole blood samples was (88.60±5.73) %,and the average CV of recovery rate was (11.70 ±5.36) %.The number of copies of C.albicans genes per unit blood was not significantly different among the same original blood samples stored separately under-20℃ for 3 or 6 months when compared with its freshly collected blood (P = 0.267).In the 74 whole blood samples obtained from the febrile surgical patients,the positive rate of C.albicans genes was 2.7% and the highest load was 4.42×103 cfu/ml.Conclusions RQ-PCR is a rapid,sensitive,highly specific,and reproducible method in detecting C.albicans NAG1 gene.Clinically it can be used to quantitatively evaluate the numbers of C.albicans in the whole blood.A small percentage of the febrile surgical patients may develop blood infection of C.albicans.

We designed the primers based on the sequence of the follistatin-related protein from Haemaphysalis longicornis Okayama strain accessed in GenBank. We cloned a gene encoding follistatin-related protein by RT-PCR, and the length cDNA is 814 bp, encoding a deduced protein of 289 amino acids. The alignment with the sequence of follistatin-related protein from the H. longicornis Okayama strain showed that the percent of nucleotide sequence and amino acid sequence is 97.8% and 99%, respectively. The expected size of GST-fused recombinant protein was 57 kD. We purified the recombinant protein through MagneGST protein purification system. Western blotting revealed that stronger reaction happened with the antiserum against eggs, but not clear with antisera against other developmental stages.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Follistatin-Related Proteins ; genetics ; immunology ; Ixodidae ; chemistry ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Alignment

Objective To explore the therapeutic effect and toxicity of Croton tiglium L.(croton)oil on rats with cold accumulation constipation,and to clarify the toxic-effect relationship of croton oil in the treatment of cold accumulation constipation.Methods The rats were orally treated with 10%2℃activated carbon solution for 3 consecutive days to replicate the model of cold constipation.The rats were fed with croton oil on the fourth day,and then the time of first defecation and the number of defecations within 3 h were observed and recorded.HE staining was used to evaluate the general pathological conditions of the colon tissues of rats.The serum levels of interleukin(IL)-6,IL-10,tumor necrosis factor(TNF)-α,cyclooxygenase(COX)-2,macrophage inflammatory protein(MIP)-1,alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total protein(TP)were detected.Immunohistochemistry was used to detect the expression of myeloid differentiation factor 88(MyD88)and nuclear transcription factor(NF)-κB P65 in colon tissues of rats.Results Croton oil could shorten the time of first defecation and increase the number of defecations within 3h in rats with cold accumulation constipation to varying degrees,and the 3.78,2.83,and 1.89 g·kg-1 croton oil group had significant statistical significance(P<0.05).HE staining results showed that compared with the model group,croton oil group could cause different degrees of inflammation and focal necrosis,goblet cell loss and lamina propria edema in the colon tissue of rats,and the 3.78 g·kg-1 croton oil group was the most serious in the colon tissue injury.In addition,compared with model group,croton oil group could increase the levels of COX-2,TNF-α and IL-6 in serum to varying degrees(P<0.05),and 3.78 g·kg-1 croton oil group had the most obvious effect.The level of MIP-1 in serum was increased in croton oil group,and the effect was significant in 3.78 g·kg-1 croton oil group(P<0.01).But croton oil group had no significant effect on serum IL-10 level of rats.The immunohistochemical results showed that the protein levels of MyD88 were increased in croton oil dose groups except 0.05 dose group,and there were significant differences in 3.78,2.83,and 1.89 g·kg-1 dose groups(P<0.05).The expression level of NF-κB p65 was enhanced in the colon of rats after croton oil treatment,and there was significant difference in 3.78 g·kg-1 croton oil group(P<0.05).In addition,compared with model group,croton dose groups had no changes in organ indexes of heart,spleen,lung,kidney and thymus(P>0.05),while liver organ indexes in1.89 and 0.94 g·kg-1 croton oil groups were significantly increased(P<0.05).The results of liver biochemical indexes showed that croton oil group did not increase the contents of ALT,AST,and TP in serum of rats,and the differences were not statistically significant(P>0.05).Conclusion Croton oil can exert a dose-dependent purgative effect at 3.78,2.83,and 1.89 g·kg-1,but it also induced colonic mucosal injury in rats by regulating the inflammatory response mediated by the MyD88/NF-κB pathway.At 0.94 and 0.47 g·kg-1,croton oil almost did not cause colon tissue injury in rats,but it had a weak purgative effect at this dose,suggesting that the best concentration range of croton oil in the treatment of cold accumulation constipation may be between 0.94 and 1.89 g·kg-1.