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Objective: To study the association of polymorphisms of FokI rs2228570 in the vitamin D receptor (VDR) gene and TMPRSS6 rs855791 with cow's milk protein allergy (CMPA) in children., Methods: Quantitative real-time PCR was used to analyze the single nucleotide polymorphisms of FokI rs2228570 in the VDR gene and TMPRSS6 rs855791 in 100 children with CMPA and 100 healthy children (control group). The multivariate logistic regression model was used to identify the risk factors for CMPA., Results: There were significant differences in the frequencies of CC, CT, and TT genotypes of TMPRSS6 rs855791 between the CMPA and control groups (P=0.008), and the CMPA group had a significantly higher frequency of TT genotype. The multivariate logistic regression analysis showed that the children with TT genotype of rs855791 had an increased risk of CMPA (OR=3.473, P=0.011). However, there was no significant difference in the genotype distribution of FokI rs2228570 in the VDR gene between the two groups (P=0.686)., Conclusions: TMPRSS6 rs855791 polymorphism is associated with CMPA in children, and TT genotype may be the susceptible genotype of CMPA. FokI rs2228570 polymorphism is not associated with CMPA.

Objective: To construct the prokaryotic expression vector of human proprotein convertase subtilisin/kexin type 9 (pcsk9), induce it to express and prepare the polyclonal antibody of human pcsk9., Methods: The sequence of pcsk9 cDNA was synthesized and amplied by PCR. By means of digestion and subcloning, the sequence was cloned into the expression vector PET-28b. The recombinant plasmid of pET-28b-pcsk9 was transformed into the competent cells of E.coli BL21 (DE3), induced to express, purified and identified. The New Zealand rabbits were immunized with the purified protein. The resulted polyclonal antibody was analyzed by ELISA and Western blotting., Results: The prokaryotic expression vector PET-28b-pcsk9 was constructed and expressed well in E.coli BL21 (DE3). We obtained the polyclonal antibody by immunizing the rabbits with the target protein. The purified antibody was proved to have a good specificity by Western blotting and its titer reached 1:13 200., Conclusion: The expression vector of pcsk9 was successfully constructed and expressed. The polycolonal antibody of pcsk9 protein was obtained as well.

Aim: To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection., Methods: The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue., Results: In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously., Conclusion: The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

Objective: To express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity., Method: Hap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay., Results: SDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05)., Conclusion: Highly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.

Objective: To determine the cocoonase content in maxilla of four silkmoth breeds of Bombyx mori and set up a dot blotting method to determine the content of the drug action components in Chinese medicines., Methods: The contents of cocoonase in maxilla of four silkworm breed were determined by dot blotting with rabbit antibody of anti-cocoonase as the first antibody. Blotting signals were analyzed by software of Quantity one., Results: Contents of cocoonase in maxilla of four silkmoth breeds were 6.41, 8.09, 7.62, and 1.88 microg/moth, respectively., Conclusion: The difference among four silkmoth breeds was significant in the content of maxilla cocoonase. Dot blotting, as a convenient and accurate method could be used for determination of the contents of drug action components in Chinese crude drugs similar to silkworm.

Hypodermin C (HC) cDNA was amplified from recombinant pGEM - T/HC, cloned in frame with the signal sequence in yeast vector pPIC9k. The plasmid was linerarized and transformed into Pichia pastoris GS115 strain by electroporation method. Recombinant strain was screened by G418 resistant, and further confirmed by PCR. The recombinant strain which contains insert was induced in the medium containing 0.5% methanol. The supernatant was collected and then purified by anion exchange chromatography. SDS-PAGE indicated that the target protein is around 28kD. Western-blot showed it can react with rabbit-anti HC serum. Gelatin substrate SDS-PAGE displayed it had enzyme activity. Provided a method to produce enough antigens for carrying out extensive immunological analyses.

In the present study,in order to investigate the pattern of the expression and location of oviductin using RT-PCR and Western blot,a 410bp cDNA fragment was amplified from mouse cervix tissue. Sequencing analysis and homology comparison with BLAST alignment showed that this fragment amplified from mouse cervix was homologous to the region from +310 to +714 of the mouse OGP gene in Genebank by the degree up to 100%. Western blot showed 60 KD and 30 KD components from mouse cervix same as those from mouse oviduct mucosa epithelium. Semi-quantitative RT-PCR analysis showed that expression of OGP was significantly higher at the estrous stage than that of non-estrous stage and almost no expression in the immature mouse. Those results suggest that the mRNA level of mouse cervix oviductin was related to estrous cycle,implied with estrogen-dependence. To further confirm the results,in situ hybridization histochemistry analysis showed that OGP mRNA was expressed at columnar epithelium of mouse cervix. To clarify the possible significance of oviductin expressed at the cervix, the function of oviductin on the mobility of human sperm was studied using "loss-of-function". The VAP, VCL, ALH, VSL showed no significant differences of the mobility of the human sperm in the conditioned medium blocked by the anti-CTP rOGP (rabbit Oviductin) antiserum compared with that in the conditioned medium of the normal rabbit serum. These results suggest that rOGP may not affect the mobility of human sperm in vitro.

Perforin and granzyme are important effector molecules in cytolytic cells. They can induce apoptosis of tumor cells and infection cells. The research of biologic missile is noticed with the progress in therapy targeting to the disease. It is supposed that perforin and granzyme play an important role in biological missiles because of their biologic function and structure. A review on several aspects of these effector molecules is presented.

Aim: To express ESP30, a novel serine protease, in Escherichia coli and to prepare anti-ESP30 antibody., Methods: ESP30 gene was amplified by PCR from the genome of aeromonas hydrophila, and cloned into the expression vector pDH2. The ESP30 expression was carried out under thermal induction. The antiserum was prepared by immunizing rabbit with ESP30. The titer and specificity of the antibody were detected by ELISA and Western blot respectively., Results: The ESP30 non-fusion protein with relative molecular mass (M(r)) of 66,000 was highly expressed in E. coli. The rabbit antibody against ESP30 was obtained. The ELISA titer of antiserum against ESP30 was about 1:128,000. Western blot analysis showed that the antiserum could bind to the expressed ESP30 specifically., Conclusion: The rabbit antibody against ESP30 has been successfully prepared, which lays the foundation for further studying the structure and function of the novel protease ESP30.

A cDNA encoding mouse implantation serine proteinase 2 ISP2 was amplified from total cDNAs of mouse uterus implantation sites on D4.5 of pregnancy by PCR, and sequenced GenBank accession No. A442918 . DNA sequencing indicated that the ISP2 cDNA had an unreported 204 bp sequence at 3' untranslated region besides the open reading frame encoding 279 amino acid residues, which was identical with literature. In order to obtain recombinant ISP2 rISP2 an expression plasmid pGEX-4T-2/ISP2 was constructed and transformed into E.coli BL21(DE3) strain. Expressed fusion protein GST-ISP2 was purified by SDS-PAGE and digested with thrombin, and the digestion mixture was subjected to SDS-PAGE again to recover rISP2. Rabbits were immunized using rISP2 as immunogen, and the polyclonal anti-ISP2 antisera were obtained. Immunohistochemical analysis using this antisera showed specific and high expression of ISP2 in mouse endometrial gland epithelium in early pregnancy.

Granzyme is an effector molecule of activated cytotoxic T cells and natural killer cells. It mainly mediates cell apoptosis. Its function could be explained by its molecular characteristics to some extent. Its cytotoxic effect is related to some other factors contributing to apoptosis induction. It deserves studying if perforin mediates entrance of granzyme into cells. As potential substrates of granzyme caspases and their substrates have been paid much attention to.