To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium, the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly. The positive clones were identified by EcoR I, Xho I digestion and sequenced. The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares. The P30 gene was recombined with plamid pET-28 a (+) to construct the prokaryotic expression vector and was expressed in E. Coli with IPTG induction. Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting. It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified, and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively. Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes. The expressed plasmid was identified by EcoR I/ Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay. It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.

Objective To develop and identify monoclonal antibodies (McAbs) against adult worm of Angiostrongylus cantonensis and observe its applicability. Methods BALB/c mice were immunized with soluble antigen of adult worms of A.cantonensis. The spleen cells of immunized mice were fused with myeloma cell, and the hybridoma secreting high titer of McAbs with high specificity was screened. By using the McAbs, serum of angiostrongyliasis patient and sera of the rats infected with A.cantonensis were detected by Western blotting and double antibody sandwich ELISA respectively. Results Three McAbs were established (2A2,3F1,4H2), which all showed no cross reaction with antigens of Schistosoma japonicum, Paragonimus westermani, Cysticercus cellulosae and Trichinella spiralis. Western blotting analysis demonstrated that the three McAbs recognized a Mr 15 000 soluble antigen of adult worm of A.cantonensis and recognized the Mr 24 000 and Mr 15 000 circulating antigens from the serum of angiostrongyliasis patient. The double antibody sandwich ELISA detection showed a positive rate of 76^5%. Conclusion Three hybridoma cell lines against adult worm of A.cantonensis have been established which secret high titer of McAbs with high specificity and seem promising in detecting the circulating antigen of the angiostrongyliasis patient.

Objective To prepare and characterize the monoclonal antibodies (McAbs) against recombinant adhesion protein 33 (AP33) of Trichomonas vaginalis. Methods The purified recombinant fusion protein AP33 was used as antigen to immunize BALB/c mice. Sp2/0 myeloma cells were fused with the splenocytes from immunized BALB/c mice. After ELISA screening and 4 times of limited dilution, 5 positive hybridoma cell lines were obtained, and the biological properties of the McAbs were identified by Western blotting. Indirect immunofluorescent antibody test (IFAT) was performed and the inhibition effect of McAbs on the cytoadherence of T.richomonas vaginalis to HeLa cell was assayed. Results Western blotting demonstrated that 5 McAbs, designated as 4A2, 4F11, 4F8, 4E7 and 4H11, specifically combined with the recombinant AP33 of T.vaginalis. The McAbs were IgG1 isotypes. Four of them (4F11, 4F8, 4E7 and 4H11) showed parasite recognition by IFAT. Parasite cytoadherence to a monolayer of HeLa cells was inhibited in vitro with a inhibition rate of 50.08%, 65.03%, 50.70% and 49.08% by the 4 McAbs under a concentration of 200, 200, 400 and 200?g/ml, respectively. Conclusions The prepared McAbs against the recombinant AP33 show a protective inhibition on cytoadherence of Trichomonas vaginalis in vitro.

Objective To investigate the polymorphisms of-139 and -336 nucleotides in dendritic cells specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) promoter region in context of HIV susceptibility, infection routines and HIV/AIDS progress. Methods Polymorphisms of -139 and -336 nucleotides in DC-SIGN were examined in 160 HIV-positive subjects and 178 healthy controls;the Spearman test was performed to analyze their associations with HIV infection status. Results In 160 HIV-positive subjects, there were 92 (57.5%) with-139C, 68 (42.5%) with-139T, 29 (18.1%) with-336C and 131 (81.9%) with -336T. The frequencies of -139T/C and -336T/C in HIV-positive subjects were similar to those in the healthy controls (χ2 =0. 121 and 1. 754, P ＞0.05 ). No differences were found in the distribution of -139T/C or -336T/C in HIV-positive subjects infected via sex intercourse or intravenous drug (χ2 =0. 435 and 0. 103, P ＞ 0. 05 ). -139C was usually companied with -336C ( r = 0. 359, P ＜ 0.01 ).-139T (27.9%) were more frequently presented in patients with CD4 +T cells ≤50 cells/μL than -139C( 23.0%, χ2 = 4.055, P ＜ 0.05 ). -139T/C and -336T/C were not related to HIV RNA levels ( t = - 0. 643and - 1. 637, P ＞ 0.05). Conclusions Genotype -139C in DC-SIGN promoter region usually coexist with -336C. Polymorphisms of -139 and -336 are not related to HIV susceptibilities or HIV infection routes.-139T genotype may be related to serious depletion on CD4 + T cells.

Cholangiocarcinoma is a group of highly invasive and heterogeneous biliary malignancies originating from any part of the biliary tree. At present, the most ideal treatment is still radical surgery.Gemcitabine combined with cisplatin (gem-cis) has been recognized as the standard first-line chemotherapy regimen for patients with unresectable, advanced or metastatic disease.In recent years, with the proposal of precision medicine and the development of next-generation sequencing technologies, A large number of important cholangiocarcinoma targets have been discovered, such as FGFR, IDH, VEGFR, BRAF, MET, etc., and the research on corresponding target drugs is booming.By referring to relevant literature and data, combined with domestic and foreign clinical trials, this paper reviews the important targets of cholangiocarcinoma and the latest progress of targeted drug therapy.

Objective:To study the difference in the extraction efficiency of the novel coronavirus (2019-nCoV) nucleic acid by using magnetic beads method, centrifugal column method and one-step method.Methods:On March 5, 2021, 10 throat swabs were collected from the staff working in the nucleic acid sampling room in Department of Clinical Laboratory, Affiliated Taikang Xianlin Drum Tower Hospital, Medical School of Nanjing University. The positive quality control samples were mixed into the swabs and used as mock positive samples. The RNA was extracted from simulated positive samples and their diluted samples by using magnetic beads method, centrifugation column method and one-step method. The purity ( A260/ A280 ratio) and concentration of the nucleic acid obtained were measured by micro-uv photometry, and fluorescence quantitative PCR was performed to compare the CT value and extraction efficiency. The three methods were used to extract the simulated weak positive specimens and to compare the difference of CT values after amplification. The measurement data that followed normal distribution were expressed by xˉ±s, the t test was used for comparing in the same group, and single factor analysis of variance was used for comparing among multiple groups. A P value smaller than 0.05 indicated a significant difference. Results:2019-nCoV nucleic acid extracted by magnetic bead method, centrifugal column method and one-step method could amplify positive results. There was no significant difference between the CT value of RNA amplification extracted by magnetic bead method and one-step method ( t=? 0.995 , P=0.376). The CT values of orf1ab gene amplified by centrifugal column method, magnetic bead method and one-step method were 29.28±0.06, 30.82±0.14 and 29.79±0.01 respectively ( F=11.196 , P=0.041). The CT values of E gene were 28.52±0.40, 27.33±0.78 and 27.38±0.13 respectively ( F=3.407, P=0.169). The CT values of N gene were 28.61±1.02, 27.24±0.20 and 27.25±0.47, respectively ( F=2.880 , P=0.020). The CT values of human genes extracted by centrifugal column method, magnetic bead method and one-step method were 19.68±0.36, 20.14±0.06 and 20.58±0.49 respectively, which was statistically significant ( F=4.904, P=0.048). The CT value of amplified human gene was affected by the dilution of human samples twice. The CT value of undiluted samples was smaller than that of diluted samples twice, with a difference of 2.95±0.22, which was statistically significant ( t=?3.025, P=0.039). The extraction time of one-step method, magnetic bead method and centrifugal column method were (15.00±1.50), (20.00±1.50) and (40.00±5.5) min respectively, and the difference was statistically significant ( F=688 , P=0.027). Conclusions:Magnetic bead method, centrifugal column method and one-step method can be used to extract 2019-nCoV nucleic acid, for the centrifugal column method has a higher extraction efficiency than the magnetic bead method and the one-step method. The one-step method is the fastest, followed by the magnetic bead method and the centrifugal column method. A large number of clinical samples can be processed using the magnetic bead method and one-step method. One-step rapid nucleic acid test can also be performed on samples from emergency and fever clinics. It is not recommended to dilute specimens for testing. In order to improve the detection rate, extracting RNA from highly suspected samples with negative initial nucleic acid test by centrifugal column method is suggested.