T cell exhaustion is associated with the risk of papillary thyroid carcinoma and can be a predictive and sensitive biomarker for diagnosis.

BACKGROUND: The incidence of papillary thyroid carcinoma (PTC) has been steadily increasing over the past decades. Hashimoto's thyroiditis (HT) is the most common autoimmune disease, and is related to the pathogenesis of PTC. Programmed death-1 (PD-1) is currently used for the treatment of PTC, but there are very few studies on the clinical value of PD-1 in the diagnosis and targeted therapy of PTC. METHODS: The expression of T, B, NK cells and PD-1 in the peripheral blood of 132 patients with PTC (PTC group), 48 patients with nodular goiter (NG group) and 63 healthy subjects (HP group) were detected by flow cytometry. The expression of plasma T3, T4, FT3, FT4, TSH, TGAb and TPO was detected by chemiluminescence immunoassay. Among 132 PTC, 49 PTC&HT and 83 PTC&noHT were included. Among 48 NG, 10 NG&HT and 38 NG&noHT were included. The expressions of programmed death- ligand1(PD-L1) in tumor tissues of PTC group and thyroid tissues of NG group, PD-1 and CD3 in tumor infiltration lymphocyte (TIL) were detected by immunohistochemistry. RESULTS: The expression of FT3, TGAb, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC and NG was significantly higher than that in the HP group. Moreover, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ expression had significant differences between the PTC group and the NG group. In addition, the expression of TGAb, TPO, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC&HT group was significantly higher than that in the PTC&noHT group. While, the expression of B cells, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC&HT group was higher than that in NG&HT group. PD-1 showed a significant correlation with PTC lymph node metastasis. CD3+PD-1+ and CD3+CD4+PD-1+ was higher in N1 stage than in N0 stage. Immunohistochemical results showed that the expression of PD-1, CD3 and PD-L1 in PTC was significantly higher than that in NG. CONCLUSIONS: T cell exhaustion might act as a biomarker for the differential diagnosis of PTC and NG. Patients with PTC&HT have obvious T cell exhaustion and increased expression of PD-1, PD-L1.Targeting the PD-1/PD-L1 pathway could be a new approach to prevent malignant transformation from HT to PTC&HT in the future.

A Sensitive and Rapid Assay for Mycoplasma hominis Detection Based on Recombinase Polymerase Amplification.

BACKGROUND: Mycoplasma hominis (MH) is an opportunistic pathogen, which often causes funisitis, spontaneous abortion, and low birth weight. However, current laboratory methods are time-consuming, labor-intensive, or require specialized laboratory instruments. Recombinase polymerase amplification (RPA) technology is a rapidly developing field because of the significance for clinical application and commercial value. Few studies have reported the use of RPA to detect MH. In this study, we developed the rapid MH detection assay, which may be potentially used as a sensitive point-of-care testing (POCT) in clinic. METHODS: Primers based on the MH 16SrRNA gene and gap gene were explored and screened out. The probe of RPA-LFD was designed based on the optimal primer and confirmed. The reaction conditions of temperature and time for RPA were optimized. The sensitivity and specificity of the analysis were explored. A total of 60 clinical specimens were used to verify the efficiency of the two methods. RESULTS: The optimal reaction conditions were determined as 15 minutes and 39°C. The sensitivity of RPA was 10-6 ng for MH, which is 100,000 times more sensitive than traditional PCR. Moreover, we observed another six non-target reproductive tract common pathogens without amplification products. Furthermore, we found that there was no significant difference between RPA and the cultivation method (p > 0.05). These two methods were in good agreement (κ = 0.938) when detecting clinical specimens. CONCLUSIONS: A new method for sensitive and rapid detection of MH based on RPA was successfully developed, which can be applied in large-scale screening and as a supplementary method to classical methods.

Regulation of Chlamydia spreading from the small intestine to the large intestine via an immunological barrier.

The obligate intracellular bacterium Chlamydia is a genital tract pathogen that can also colonize the gastrointestinal tract for long periods. The long-lasting colonization is dependent on chlamydial spreading from the small intestine to the large intestine. We previously reported that a mutant Chlamydia was able to activate an intestinal barrier for blocking its own spreading to the large intestine. In the current study, we used the mutant Chlamydia colonization model to confirm the intestinal barrier function and further to determine the immunological basis of the barrier with gene-deficient mice. Recombination activating gene 1-/- mice failed to block the mutant Chlamydia spreading, while mice deficient in toll-like receptors, myeloid differentiation primary response 88 or stimulator of interferon genes still blocked the spreading, suggesting that the intestinal barrier function is dependent on lymphocytes that express antigen receptors. Mice deficient in CD4, but not CD8 nor µ chain failed to prevent the chlamydial spreading, indicating a protective role of CD4+ cells in the intestinal barrier. Consistently, adoptive transfer of CD4+ T cells reconstituted the intestinal barrier in CD4-/- mice. More importantly, CD4+ but not CD8+ T cells nor B cells restored the intestinal barrier function in recombination activating gene 1-/- mice. Thus, CD4+ T cells are necessary and sufficient for maintaining the intestinal barrier function, indicating that the spread of an intracellular bacterium from the small intestine to the large intestine is regulated by an immunological barrier. This study has also laid a foundation for further illuminating the mechanisms by which a CD4+ T cell-dependent intestinal barrier regulates bacterial spreading in the gut.

Chlamydia Deficient in Plasmid-Encoded pGP3 Is Prevented from Spreading to Large Intestine.

The cryptic plasmid pCM is critical for chlamydial colonization in the gastrointestinal tract. Nevertheless, orally inoculated plasmid-free Chlamydia sp. was still able to colonize the gut. Surprisingly, orally inoculated Chlamydia sp. deficient in only plasmid-encoded pGP3 was no longer able to colonize the gut. A comparison of live organism recoveries from individual gastrointestinal tissues revealed that pGP3-deficient Chlamydia sp. survived significantly better than plasmid-free Chlamydia sp. in small intestinal tissues. However, the small intestinal pGP3-deficient Chlamydia sp. failed to reach the large intestine, explaining the lack of live pGP3-deficient Chlamydia sp. in rectal swabs following an oral inoculation. Interestingly, pGP3-deficient Chlamydia sp. was able to colonize the colon following an intracolon inoculation, suggesting that pGP3-deficient Chlamydia sp. might be prevented from spreading from the small intestine to the large intestine. This hypothesis is supported by the finding that following an intrajejunal inoculation that bypasses the gastric barrier, pGP3-deficient Chlamydia sp. still failed to reach the large intestine, although similarly inoculated plasmid-free Chlamydia sp. was able to do so. Interestingly, when both types of organisms were intrajejunally coinoculated into the same mouse small intestine, plasmid-free Chlamydia sp. was no longer able to spread to the large intestine, suggesting that pGP3-deficient Chlamydia sp. might be able to activate an intestinal resistance for regulating Chlamydia sp. spreading. Thus, the current study has not only provided evidence for reconciling a previously identified conflicting phenotype but also revealed a potential intestinal resistance to chlamydial spreading. Efforts are under way to further define the mechanism of the putative intestinal resistance.

Chlamydia pneumoniae inclusion membrane protein Cpn0147 interacts with host protein CREB3.

Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impacts. Like other chlamydial species, Chlamydia pneumoniae possesses a unique developmental cycle, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. The cytoplasmic vacuole where Chlamydia pneumoniae replicates is called an inclusion, which is extensively modified by the insertion of chlamydial effectors known as inclusion membrane proteins (Incs). The C. pneumoniae-specific inclusion membrane protein (Inc) Cpn0147 contains domains that are predicted to be exposed to the host cytoplasm. To map host cell binding partners of Cpn0147, a yeast two-hybrid system was used to screen Cpn0147 against a HeLa cell cDNA library, which led to the finding that Cpn0147 interacted with the host cell protein cyclic adenosine monophosphate (cAMP)-responsive element (CRE)-binding protein (CREB3). The interaction was validated by co-immunoprecipitation of Cpn0147 with CREB3 from HeLa cells ectopically expressing both. Furthermore, Cpn0147 and CREB3 were co-localised in HeLa cells under confocal fluorescence microscopy. The above observations suggest that CREB3 may directly bind to the cytoplasmic domain of Cpn0147 to mediate the interactions of chlamydial inclusions with host cell endoplasmic reticulum.

[Expression of mannose-binding lectin associated protein 19 (MAp19) in HeLa cells].

OBJECTIVE: To construct an eukaryotic expression vector of mannose-binding lectin associated protein 19 (MAp19) and further express MAp19 fusion proteins. METHODS: MAp19 gene fragment was amplified by PCR. The eukaryotic expression vector pcDNA3.1/Myc-His A-MAp19 was constructed by gene cloning technology, identified by restriction enzyme digestion and further confirmed by sequencing. The recombined plasmid was then transfected into HeLa cells by liposome-mediated method, and the positive clones with vector pcDNA3.1/Myc-His A-MAp19 were selected by G418. Location of MAp19 fusion proteins in the transfected HeLa cells was observed under the fluorescence microscope, and the expression levels of MAp19 fusion proteins were detected by Western blotting. RESULTS: The recombined vector pcDNA3.1/Myc-His A-MAp19 was successfully constructed. The result of DNA sequencing was in accordance with NCBI data bank. By G418 selecting, we obtained the HeLa cells which could stably express exogenous MAp19 fusion proteins. The protein was located in the cytoplasm. Western blotting also suggested that the MAp19 was secretory protein. CONCLUSION: The vector expressing MAp19 has been prepared successfully, and can express the target protein (MAp19) in the eukaryotic cells (HeLa cells).

MicroRNA­1915­3p prevents the apoptosis of lung cancer cells by downregulating DRG2 and PBX2.

Micro (mi)RNAs are short non­coding RNA molecules, which post­transcriptionally regulate gene expression and exert key roles in cell growth, differentiation and apoptosis. In the present study, the mechanism and the function of miR­1915­3p in the apoptotic regulation of lung cancer cell lines (NCI­H441 and NCI­H1650) were investigated. The expression analysis confirmed that the expression of miR­1915­3p was markedly decreased in the apoptotic cells. The overexpression of miR­1915­3p in the lung cancer cells prevented apoptosis induced by etoposide. Developmentally regulated GTP­binding protein 2 (DRG2) and pre­B cell leukemia homeobox 2 (PBX2) were identified as downstream targets of miR­1915­3p, which was shown to bind directly to the 3'­untranslated region of DRG2 and PBX2, subsequently lowering their mRNA and protein expression levels. Co­expression of miR­1915­3p and DRG2/PBX2 in the NCI­H441 and NCI­H1650 cells partly circumvented the effect of miR­1915­3p on apoptosis. The results in the present study revealed that miR­1915­3p functions as a silencer of apoptosis, which regulates lung cancer apoptosis via targeting DRG2/PBX2, and consequently this miRNA may be a putative therapeutic target in lung cancer.

[Construction of Eukaryotic Expression Vector Containing ROP18-ROP12 of Toxoplasma gondii RH Strain].

OBJECTIVE: To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12 (encoding rhoptry protein 18 and 12) complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells. METHODS: Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamH I and Xba I . ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18- ROP12. After colony PCR, enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene ß-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein. RESULTS: Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by Hind III, BamH I and Xba I digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene (Accession No. AM075204.1) and 99% identical to that of T. gondii RH strain ROP12 gene (Accession No. DQ096559.1). Further, RT-PCR showed amplification products at 613 bp for ß-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2,373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12. CONCLUSIONS: The recombinant eukaryotic plasmid pVAX1-ROP18-ROP-2 is constructed and can be expressed in eukaryotic system.

[Measurement of oxygen concentration using multimode diode laser absorption spectroscopy].

Tunable diode laser absorption spectroscopy (TDLAS) is a widely used technique for high sensitivity, good selectivity and fast response. It is widely used in environment monitoring, industrial process control and biomedical sensing. In order to overcome the drawbacks of TDLAS including high cost, poor stability and center wavelength shift problem. A multi-mode diode laser system based on correlation spectroscopy and wavelength modulation spectroscopy (TMDL-COSPEC-WMS) was used to measure O2 concentration near 760nm at the 1%~30% range of near room temperature. During the experiment, the light is splitter into two beams, respectively through the sample and measuring cell, two receiving optical signal collection containing gas concentration information sent back stage treatment, invert the oxygen concentration through correlation and ratio between measured signal and reference signal, the correlation spectroscopy harmonic detection technique is used to improve the stability of the system and the signal to noise ratio. The result showed that, there was a good linear relationship between the measured oxygen concentration and the actual concentration value. A detection limit of 280 pmm. m in the 1 atmospheric which approved of the same sample. A continuous measurement for oxygen with the standard deviation of 0. 056% in ambient air during approximately 30 minutes confirms the stability and the capability of the system. The design of the system includes soft and hardware can meet the needs of oxygen online monitoring. The experimental device is simple and easy to use, easy to complex environment application.

[Preparation and identification of polyclonal antibodies against CUB1 and CUB2 functional domains from mannan-binding lectin-associated serine protease-2].

OBJECTIVE: To express CUB1 and CUB2 functional domain proteins of mannan-binding lectin-associated serine protease-2 (MASP-2) by prokaryotic expression system, and further prepare and identify the polyclonal antibodies against these two domains. METHODS: The pGEX-6P-2 prokaryotic vector carrying the target gene CUB1 and CUB2 was used to prepare the recombinant protein GST-CUB1 and GST-CUB2. These two fusion proteins were purified with Glutathione SepharoseTM4B beads and then combined with Freund's adjuvant as antigens to immunize the BALB/c female mice aged 5 weeks for generating polyclonal antibodies. Subsequently, the specificity of the polyclonal antibodies was detected by Western blotting, and the titer was determined by indirect ELISA. RESULTS: The fusion proteins GST-CUB1 and GST-CUB2 were successfully expressed and purified. Their polyclonal antibodies were gained from the BALB/c mice immunized with these two proteins. The polyclonal antibodies showed high specificity with no cross-reaction with other proteins. Indirect ELISA indicated that the titer of anti-CUB1 antibody was more than 1:32 000 and anti-CUB2 antibody was more than 1:16,000. CONCLUSION: The polyclonal antibodies against GST-CUB1 and GST-CUB2 fusion proteins were obtained respectively with high titers and strong specificity.

[HLA-E gene polymorphisms and plasma soluble HLA-E levels and their relationship with genetic susceptibility to breast cancer].

OBJECTIVE: To investigate the association of human leukocyte antigen-E (HLA-E) gene polymorphisms and plasma soluble HLA-E (sHLA-E) levels with genetic susceptibility to breast cancer in Zhangjiakou women of Han nationality. METHODS: HLA-E typing was performed in 200 patients with breast cancer and 228 healthy controls from Zhangjiakou area by means of polymerase chain reaction-sequence-specific priming (PCR-SSP). Simultaneously, sHLA-E was detected by ELISA. RESULTS: We found two alleles of HLA-E: HLA-E*0101 and HLA-E*0103, and three genotypes, namely HLA-E*0101/HLA-E*0101, HLA-E*0101/HLA-E*0103 and HLA-E*0103/HLA-E*0103. The frequencies of HLA-E*0103 allele and HLA-E*0103/HLA-E*0103 genotype showed a significant difference between healthy controls and patients with breast cancer. The HLA-E*0103/HLA-E*0103 genotype significantly increased the risk for breast cancer. Plasma levels of sHLA-E showed no significant difference in breast cancer patients and controls. While in breast cancer patients with HLA-E*0103/HLA-E*0103 genotype, plasma levels of sHLA-E were significantly elevated compared with those in healthy controls. HLA-E genotypes and sHLA-E levels had no obvious relationship with tumor grading. CONCLUSION: HLA-E gene polymorphisms are associated with the development of breast cancer in Zhangjiakou women of Han nationality. HLA-E*0103/HLA-E*0103 genotype is probably susceptible genotype for breast cancer, while plasma sHLA-E level probably has no association with breast cancer susceptibility.

[Cloning and bioinformatics analysis of rhoptry protein 11 of Toxoplasma gondii].

Total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii. The open reading frame of ROP11 gene was amplified by using a pair of specific primers designed according to the coding sequence of ROP11 gene (Accession No. DQ077905). The RT-PCR product was digested by restriction enzyme EcoR I and Not I, and then ligated into a pGEX-6P-2 vector. The recombinant plasmid was transferred into E. coli XL-Blue. The positive clones was selected by colony PCR, and confirmed by the double restriction enzyme digestion and sequencing. The RT-PCR product was 1,548 bp. The recombinant plasmid was confirmed by colony PCR and double restriction enzyme digestion. Sequencing results showed that the obtained ROP11 gene was 1 548 bp (Accession No. KC456639). There was a high sequence consistency (99%) between the obtained ROP11 gene sequence and the Toxoplasma ROP11 gene from GenBank. Bioinformatics analysis showed that the ROP11 protein (Mr 57,020) consisted of the signal peptide (amino acids 1-26), 12 conservative domains, a serine/threonine protein kinase catalytic domain (amino acids 170-511), and two potential N-glycosylation sites.

[Expression of EGF and SP domain proteins of MASP-2 and preparation of their polyclonal antibodies].

AIM: To clone and prokaryotically express the EGF and SP domain proteins of human mannan-binding lectin associated serine protease-2 (MASP-2), and prepare their polyclonal antibodies and analyze their immunogenicity. METHODS: The cDNA of human MASP-2 was transcripted reversely from total RNA extracted from human fetal liver tissue, and then EGF and SP domain genes of MASP-2 were amplified from the cDNA by PCR. The EGF and SP fragment domain genes were cloned into pGEX-6P-2 expression vector and induced by IPTG to express GST fusion protein. The BALB/c mice were immunized with the purified fusion proteins to generate polyclonal antibodies. The specificity of the polyclonal antibodies was analysed with Western blotting, and the titer was determined by indirect ELISA. RESULTS: We successfully constructed the recombinant pGEX-6P-2-EGF and pGEX-6P-2-SP and induced the expressions of the GST-EGF and GST-SP fusion proteins. The specificity of the polyclonal antibodies was higher, and had no cross reaction with other proteins. Indirect ELISA showed that the titer of anti-EGF antibody was more than 1:32 000 and anti-SP antibody was more than 1:40 000. CONCLUSION: The polyclonal antibodies of the EGF and SP domain proteins we successfully obtained were of higher specificity and titer, which provides the basis for further research on MASP-2.

[Cloning, expression and immunoreactivity analysis of rhoptry protein 18 (rop18) from Toxoplasma gondii].

OBJECTIVE: To clone and express rhoptry protein 18 (ROP18) gene of Toxoplasma gondii, and analyze its immunoreactivity. METHODS: The genomic DNA was extracted from T. gondii (RH strain) tachyzoites. TgROP18 gene was amplified by PCR, and cloned into pET30a (+) vector. The constructed pET30a (+)-TgROP18 was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed through SDS-PAGE, and identified by Western blotting with mouse anti-T. gondii serum. RESULTS: The TgROP18 gene was about 1 665 bp in length and encoded for a protein of 544 amino acid residues and the former 47 amino acids consisted signal peptide sequences. PCR, enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET30a (+)-TgROP18 was constructed. Bacteria containing recombinant plasmid pET30a (+)-TgROP18 expressed a soluble protein of His-TgROP18 (M, 59 800) after being induced with IPTG. His-TgROP18 reacted positively with mouse anti-T. gondii serum by Western blotting analysis. CONCLUSION: The soluble His-TgROP18 protein shows certain immunoreactivity.

[Establishment of loop-mediated isothermal amplification (LAMP) for detecting Pneumocystis carinii].

Animal model of Pneumocystis carinii pneumonia (PCP) was established for acquiring lung tissue infected with P. carinii. After DNA from rat lungs was extracted, nuclear ribosome small subunit 18s rDNA of P. carinii was amplified by loop-mediated isothermal amplification method at 63 degrees C for 60 min. The product was digested by restriction enzyme Apal I. The results showed that 18s ribosome DNA (rDNA) of P. carinii was cloned into vector pGEX6p2, and the positive clones screened. Therefore, the loop-mediated isothermal amplification has been established for detecting P. carinii.

Association between mannose-binding lectin and HIV infection and progression in a Chinese population.

Mannose-binding lectin (MBL) is a circulating pattern recognition molecule involved in the innate immune system that mediates phagocytosis and activates complement by binding to a carbohydrate extremity. Some MBL genetic polymorphisms result in deficient protein levels and increased susceptibility to infection. The objective of this study was to investigate the association between MBL2 exon 1 polymorphisms, serum levels of normal MBL, and HIV-1 infection and progression in a Chinese population. A total of 1075 adult patients infected with HIV-1 (532 male and 543 female) were recruited. The genotype of 145 patients was determined and the genotype frequencies compared with healthy population controls. The disease status of patients was evaluated for different MBL2 genotypes and normal MBL serum levels. MBL2 exon 1 polymorphisms (A/O or O/O) were significantly more common in HIV-1-infected patients than in the healthy controls. Patients in clinical categories B/C with severe diseases were significantly more likely to have one variant allele. There was a statistical relationship between MBL2 genotypes and MBL serum levels. In addition, higher CD4(+) T cell counts and ratios of CD4(+) T cells:CD8(+) T cells were observed in patients with medium MBL levels than with low or high MBL levels. Patients with mild diseases were also more likely to have medium MBL levels than high levels. Analysis of MBL levels with respect to sex yielded differences. Median MBL levels were substantially higher in males than in females in HIV-1-infected patients. Lower CD4(+) T cell counts were detected in males with low MBL levels, but the opposite was observed in females. Our results suggest that genetic polymorphisms resulting in MBL deficiency are associated with increased susceptibility to HIV-1 infection and disease progression in the studied population. Moreover, serum circulating levels of normal MBL in HIV-1-infected patients could be an important auxiliary biological marker in association with CD4(+) T cell counts in the evaluation of HIV-1 disease progression. The sex differences in the regulation of MBL serum levels during infection merit further exploration.

[Study on gene cloning of Chlamydial pneumonia CPn0308 and its endogenous localization].

OBJECTIVE: To clone CPn0308 gene from Clamyida pneumonia and express its fusion protein, to make antibodies to fusion protein GST-CPn0308, and to further localize endogenous protein preliminarily using antibodies raised with CPn0308 fusion protein. METHODS: The open reading frame (ORF) coding for CPn0308 in the Chlamydia pneumonia AR 39 genome was cloned into the pGEX6p2 vector after it was cloned using PCR and digested by the restriction enzymes BamHI and NotI. The recombinant plasmid pGEX6p2-CPn0308 was transformed into XL1-blue bacteria and the gene CPn0308 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CPn0308 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CPn0308 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). RESULTS: The CPn0308 gene, which was 366bp in length,was successfully cloned and the GST fusion protein with molecular weight of 39kD was expressed. It was found that the hypothetical protein CPn0308 was located in the inclusion membrane of Chlamydia pneumonia-infected cells using IFA of mouse anti-fusion protein antibodies. CONCLUSIONS: Using antibodies raised with GST-CPn0308 fusion protein, the hypothetical protein CPn0308 was identified to be a Chlamydia pneumoniae inclusion membrane protein. It could be the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells.

[Enzyme change in bronchoalveolar lavage fluid of pneumocystis pneumonia rats and the effect of garlicin treatment].

OBJECTIVE: To study the change of enzymes and effect of garlicin treatment on the change in bronchoalveolar lavage fluid (BALF) of rats with Pneumocystis carinii pneumonia (PCP). METHODS: Wistar rats were injected intramuscularly continually with dexamethasone to establish the rat model of PCP. The experimental rats (group A) were injected intramuscularly with garlicin at a dose of 10 mg/(kg x d) for 5 days in the 3rd, 6th and 9th week respectively, and SMZ/TMP therapy group (B), PCP infected group (C) and normal group (D) were established as controls. Three days after the last treatment, the rats of all groups were killed and BALF was collected without contamination and enzymes AST, ALF, CHE, ALP, LDH, CK, CKMB, HBDH, AFU, 5'NT, ADA were examined. RESULTS: The ALP level in group C [(573.41 +/- 350.63)U/L] was significantly higher than that in group D [(210.56 +/- 114.41) U/L] (q = 4.682, P < 0.01), group A [(392.07 +/- 217.57) U/L] (q = 3.851, P < 0.05), and group B [(325.21 +/- 180.65) U/L] (q = 4.380, P < 0.01); the level of CK, CKMB and 5'NT in group C [948.94 +/- 403.43, 489.47 +/- 254.46 and (6.76 +/- 3.11) U/L respectively] was higher than those in group D [426.22 +/- 319.00, 213.33 +/- 144.54 and (3.22 +/- 1.20) U/L] (q = 4.696, 3.784, 3.812, P< 0.05); there was no significant difference in the level of AST, ALT, CHE, LDH, HBDH, AFU and ADA among the four groups (F = 1.852, 0.958, 2.470, 1.423, 1.178, 1.342, 0.611, P > 0.05). CONCLUSIONS: The level of ALP, CK, CKMB but the ALP level decreases distinctly after the garlicin and 5'NT increases evidently in BALF of PCP infected rats, but the ALP level decreases distinctly after the garlicin treatment.

[Localization of the hypothetical protein CT249 in the Chlamydia trachomatis inclusion membrane].

To characterize the hypothetical protein CT249 using antibodies raised with CT249 fusion protein. The open reading frame (ORF) coding for CT249 in the Chlamydia trachomatis serovar L2 genome was cloned into the pGEX-6p2 vector using the restriction enzymes BamH I and Not I. The recombinant plasmid pGEX-6p2-CT249 was transformed into XL1-blue bacteria and the gene CT249 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CT249 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CT249 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). The CT249 gene with 351bps in length was successfully cloned and expressed as GST fusion protein with molecular weight of 38.2kDa. The anti-fusion protein antibodies produced from mice detected the hypothetical protein CT249 in the inclusion membrane of Chlamydia trachomatis-infected cells. Using antibodies raised with GST-CT249 fusion protein, the hypothetical protein CT249 have been identified as a Chlamydia trachomatis inclusion membrane protein. Given the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells, this finding has provided a useful tool for further understanding the mechanisms of chlamydial intracellular parasitism.