Hepatitis B virus induces expression of cholesterol metabolism-related genes via TLR2 in HepG2 cells

AIM:To investigate whether hepatitis B virus(HBV) exacerbates hepatic cholesterol accumulation,and explore the underlying mechanisms.METHODS:HepG2 cells were infected with adenovirus(Ad) containing 1.3-fold overlength HBV genome.Realtime polymerase chain reaction and Western blotting were used to measure mRNA and protein expression of target genes.Cholesterol accumulation was measured by fluorescence microscopy.Cell toxicity due to Ad-HBV treatment was determined by the mitochondrial tetrazolium assay.The protein levels of toll-like receptors(TLRs) were determined by Western blotting.RESULTS:Ad-HBV increased hepatic cholesterol accumulation and enhanced the mRNA and protein levels oflow-density lipoprotein receptor(LDLR) and 3-hydroxy3-methylglutharyl-coenzyme A reductase(HMGCoAr) mRNA and protein expression in HepG2 cells.In addition,these inductive effects were partly offset by suppressing TLR2 expression levels by small interfering RNA in HepG2 cells.CONCLUSION:Ad-HBV increases LDLR and HMGCoAr expression,resulting in exacerbated cholesterol accumulation in HepG2 cells,which was mediated via the TLR2 pathway.

A Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China

Objective To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. Methods FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and irnmunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples （159 patients with Lyme disease and 292 controls）, all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

Construction of the Eukaryotic Expression Vector for Outer Membrane Protein Tp92 from Treponema pallidum and Its Preliminary Study on the Immune Responses in New Zealand Rabbits

To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.

Eukaryotic expression of outer membrane protein Gpd from Treponema pallidum and preliminary studies on its immune response in rabbits

The Gpd gene was amplified from the genomic DNA of Treponema pallidum and cloned into the appropriate site of pcDNA3.1(+) vector. The expression of pcDNA3.1(+)-Gpd in HeLa cells was tested with Western blotting and technology of immunocytochemistry. New Zealand rabbits were immunized with the eukaryotic expression recombinant pcDNA3.1(+)-Gpd. A fusion protein of Gpd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected by Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1∶1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls (P<0.01). All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.

Preparation and analysis of immunocompetence of recombinant fusion protein of the immunodominant region in chlamydial protease-like activity factor from Chlamydophila pneumoniae and its application in serodiagnosis

To clone the gene coding the immunodominant region in the chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,to analyze immunocompetence of the expressed protein, and to evaluate its value in serodiagnosis,the CPAF immunodominant region gene was amplified,ligated into a pGEX6p-2 vector,and then the expressed recombinant protein was purified with glutathione S- transferase(GST)agarose gel FF after renaturation,then identified by SDS-PAGE and Western blot.A new indirect ELISA was developed with the purified protein as coating antigen.The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits,and its immunoreactivity was analyzed by reacting with anti-C,pneumoniae antibody.300 clinical sera samples were respectively de- tected by microimmunofluorescence(MIF)as reference method and the indirect ELISA,and the differ- ence between the two methods was analyzed.Cross-reactivity against Chlamydia trachomatis was investi- gated with the indirect ELISA to detect anti-C,trachomatis positive antisera.The results indicated that a 51.3 kDa recombinant protein was obtained.Western blot assay proved that the recombinant protein could merely specifically react with human anti-C.pneumoniae antisera.The titers of the specific IgG an- tibodies in the immunized New Zealand rabbits were above 1:16 000.Anti-C.pneumoniae IgG positive and negative reference sere were detected with the indirect ELISA,and the concordance rate of negative and positive results were both 100%(40/40).The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8%(45/48)and 100%(252/252)separately by detecting 300 clinical sera samples,and the concordance rate between the two methods was 99.0%.No cross reaction against C.trachomatis was found with the indirect ELISA to detect anti-C,trachomatis positive antisera.In con- clusion,the prepared recombinant protein of the CPAF immunodominant region shows excellent immuno- competence and can be used to develop a new indirect ELISA as a method to detect anti-C.pneumoniae antibody for diagnosis of C.pneumoniae infection.

Effects of the polysaccharides from Spirulina platensis on the activities of hepatitis B virus

To investigate the effects of the polysaccharides from Spirulina platensis (PSP) on hepatitis B virus (HBV), the cytotoxic effect of PSP was assessed by choosing the maximal concentration of PSP without cytotoxic effect on HepG2 2.2.15 cell line for further experiments. Four concentrations (400-50 μg/ml) of PSP were adopted to treat the cells, and the supernatants or cells were collected after 24, 48, 72 and 144 h respectively. HBsAg and HBeAg in the supernatants of cell cultures were tested with ELISA and copies of HBV DNA in supernatants were measured by real-time fluorescence quantitative PCR (FQ-PCR). Meanwhile, DNA/RNA hybridization was performed to evaluate the expression of IFN-α receptor (IFN-αR) on the cells. The experimental results showed that the secretion of HBV antigens decreased under the influence of PSP in a dose and time-dependent manner. PSP in concentration of 400 μg/ml could significantly decrease the secretion of HBsAg in 24 h. Although no obvious effect was observed on the secretion of HBeAg at that time, the inhibitory effects were observed in a dose-dependent manner from 48 to 144 h. In addition, the copies of HBV DNA were declined under the influence of PSP in the same manner, moreover, the maximal suppressive effect of PSP in concentration of 400 μg/ml was as great as that of lamivudine. The expression of IFN-αR was much higher in PSP-treated cells than that of the un-treated cells also in dose and time-dependent manner. It is concluded that PSP in non-cytotoxic concentration not only significantly decreases the secretion of the HBV antigens and the replication of HBV DNA, but also increases the expression of IFN-αR on HepG2 2.2.15 cell line. The results of the present investigation strongly support the notion that PSP exerts the anti-HBV effect both through enhancing the anti-HBV immunity and acting on HBV directly.

Characterization of Chlamydia trachomatis omp1 gene among sexually transmitted disease patients in south China

To investigate the DNA sequence polymorphism of Chlamydia trachomatis omp1 gene, urogenital samples were collected from 4 different cities in South China, DNA was extracted, and an approximately 980-bp-long fragment of the omp1 gene was amplified by nested polymerase chain reaction (nPCR). DNA sequence was determined, genotyping was performed by BLAST similarity search, and multiple alignment was performed with CLUSTAL X. Then a phylogenetic tree was constructed by Mega 3 software to illustrate the evolutionary relationships between clinical isolates and reference strains. Ninety-six specimens were sequenced, and 28 genetic variants were detected, among which E was the most prevalent genotype. The omp1 gene was highly conserved for genotypes E and F, but appeared slightly less conserved for other genotypes, where the sequences displayed one to several nucleotide substitutions relative to the reference sequence. Phylogenetic tree showed that C.trachomatis serotypes were mainly divided into three clusters, according to previous grouping in the B, F-G, and C complexes, and the clinical isolates were highly related to the corresponding reference strains. It concluded that the omp1 gene of the isolated C.trachomatis strains exhibited remarkable DNA sequence polymorphism, which can encourage for vaccine design and infection control.

Effect of HCMV IE1 Protein on Cytokines Secretion and Apoptosis of Macrophages

Objective： To investigate the effect of human cytomegalovirus （HCMV） IE1 protein on the secretory activity and apoptosis of macrophages. Methods： The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect THP-1-macrophages. 48 h after transfection, the expression and localization of GFP or GFP-IE1 was observed under fluorescent microscope. The levels of IL-1β and TNF-α in the culture media were examined by ELISA, and the mRNA expression of them was analyzed by RT-PCR. Cell undergoing apoptosis were determined by flow cytometry using the propidium iodide （PI） staining method. The data were analyzed by SPSSI3.0. Results： As observed under fluorescent microscope, the expressions of GFP-IEI and GFP by plasmid pEGFP-C1/IE1 or pEGFP-C1 in THP-1-macrophages could be found in nuclei or whole cells. Conclusion： As demonstrated by RT-PCR and ELISA, mRNA and protein expressions of IL-1β and TNF-α and promotes apoptosis in THP-1-macrophages.

Hepatitis B Virus X Protein Up-regulates TNF-α and IL-1β Secretion of Macrophages

Objective： To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus （HBV） X protein （HBx） on hepatocellular carcinoma. Methods： Reconstructed plasmid pcDNA3.1（＋）-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results： In THP-lmacrophages, the pcDNA3.1（＋）-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following： control group was different from LPS group and pcDNA3.1（＋） group （P〈0.01）, and so was pcDNA3.1（＋）-HBx group; but there was no obvious difference between pcDNA3.1（＋） group and LPS group （P〉0.05）, all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages. Conclusion： Transient overexpression of HBx up-regulates LPS-induced TNF-t~ and IL-113 secretion of macrophages.

The Construction of the Eukaryotic Expression Vector of Glycerophosphodiester Phosphodiesterase Gene from Treponema pallidum and its Expression in Hela Cells

Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.

Transient over-expression of human papillomavirus type 16 E6 protein down-regulate the secretion of TNF-αor IL-1β LPS-induced from macrophages

In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16(HPV16),the eukaryotie expression vector pcDNA3.1(-)/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-indueed THP-1-macrophages,and the reconstructed plasmid pcDNA3.1(-)/E6 was transfected into THP-1-maerophages.The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-αor IL-1βwas examined by ELISA.All of data were analyzed by SPSS12.0.As demonstrated by Western blot analysis,the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1(-)/E6 in THP-1-macrophages could be detected.Howev- er,as demonstrated by ELISA assay,the level of TNF-αor IL-1βin lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1(-)/E6 group and the LPS control group or the pcDNA3.1(-)control group(P<0.01),but no significant difference existed between pcDNA3.1(-) control group and LPS control group(P>0.05).All these results illustrate that the transient over-ex- pression of HPV6 E6 protein reduces the production of TNF-αand IL-1βinduced by LPS in THP-1-mac- rophages.

Expression and functional identification of the hypothetical adhesin P32 from Mycoplasma genitalium

Mycoplasma genitalium is the main causative agent for non-gonococcal and non-chlamydial urethritis. P32 is the putative surface-exposed membrane protein of M. genitalium and it has substaintial identity in amino acid sequence with adhesin protein P30 from M. pnewnoniae. Since M. pneumoniae mutants lacking P30 protein is defective in cytadherence, P32 protein has been proposed to be an essential adhesin implicated in the adherence of M. genitalium to host cells. The prokaryotic expression vector pET-30 ( + )/p32 was constructed in the present study, and the recombinant protein was expressed in E. coli and purified under denaturing condition. As demonstrated by the immunoblotting analysis, the recombinant protein could react with rabbit antisera against M. genitalium, and adherence inhibition assays were petformed with antisera against this recombinant protein. It was demonstrated that P32 protein apperared to be an adhesion protein of M. genitalium, thus providing the experimental basis for better understanding of the pathogenesis of M. genitalium infection and for the development of the related vaccines against the infection.

Expression of inducible nitric oxide synthase induced by lipid-associated membrane proteins of Ureaplasma urealyticum is regulated by nuclear factor κB-mediated mechanism in murine macrophages

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase (iNOS) gene expression stimulated by lipid associated membrane proteins (LAMPs) of Ureaplasma urealyticum (U.urealyticum). Detection of NO, the expression of iNOS and the activation of nuclear factor κB (NF-κB) in direct response to U.urealyticum LAMPs in a murine macrophages, the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB and of cycloheximide (CHX), a protein synthase inhibitor were available. The results indicated that U.urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manner by activating NF-κB. The expression of iNOS, NO production and the activation of NF-κB were inhibited by U.urealyticum LAMPs combination with PDTC or CHX. In conclusion, our findings suggest that U.urealyticum may be an etiological factor to certain diseases due to its ability to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.

Expression and purification of Ctomp2aa167-aa434 and preparation of its mAb

Chlamydia trachomatis outer membrane protein 2 (Ctomp2) is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species . To purify the protein and to prepare monoclonal antibodies (mAbs) against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni2+ -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1:40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans . It had high specifity. In comparison with gold standard test-cell culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein , but also to the establishment of the method for its clinical application, for it had not been reported before.

Interactions between mycoplasma lipid-associated membrane proteins and the host cells

Mycoplamas are a group of wall-less prokaryotes widely distributed in nature, some of which are pathogenic for humans and animals. There are many lipoproteins anchored on the outer face of the plasma membrane, called lipid-associated membrane proteins (LAMPs). LAMPs are highly antigenic and could undergo phase and size variation, and are recognized by the innate immune system through Toll-like receptors (TLR) 2 and 6. LAMPs can modulate the immune system, and could induce immune cells apoptosis or death. In addition, they may associate with malignant transformation of host cells and are also con-sidered to be cofactors in the progression of AIDS.

HPV16 E6蛋白与hDaxx的相互作用及其对HeLa细胞凋亡的影响(英文)

Objective: To study the interaction of human papillomavirus type 16 (HPV16 E6) protein and human death domain associated protein (hDaxx) and its effect on apoptosis of HeLa cells to provide the experimental basis for exploring the oncogenic mechanism of HPV16 E6 protein. Methods: Recombinant vector of pGADT7/E6 or pGBKT7/hDaxx was con- structed. The interaction of E6 protein and hDaxx was detected by yeast two-hybrid system. Their expression in yeast was detected by Western blotting. The eukaryotic plasmids of E6 and hDaxx were co-transfected into HeLa cells. Apoptosis was induced by 5-FU. The apoptotic rate was measured by flow cytometry (FCM). Results: E6 protein had intracellular interaction with hDaxx. The apoptotic rate was rising with the increase in the transfection quantity of pcDNA3.1 (-) / hDaxx in pcDNA3.1 (-) /E6 and pcDNA3.1 (-) / hDaxx co-transfected cells. The difference was significant ( P < 0. 01). Conclusion: There is intracellular interaction between HPV16 E6 protein and hDaxx. The over-expression of hDaxx can increase the sensitivity of E6 protein positive HeLa cells to 5-FU. The effect was in a dose dependent manner. HPV16 E6 protein inhibited the apoptosis of HeLa cells by interacting with hDaxx.

Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China

Background Mycobacterial interspersed repetitive units-variable number tandem repeat （MIRU-VNTR） and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium （M.） tuberculosis. We collected 218 strains of M. tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China. Methods MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains, and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region. Results The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates （55.05%）; the cluster rate was 47.25%. By detecting the deletion of RD105 sequence, 188 of 218 （86.23%） isolates belonged to Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates （54.13%）. The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family. Conclusions The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu. Combining detecting the deletion of RD105 and MIRU typing together provides a simple, fast, and effective method which is low in cost and might be practical and suitable for M. tuberculosis aenotvDina in China.

Transcriptional repression of hDaxx enhanced by adenovirus 12 E1B 55-kDa oncoprotein interacting with hDaxx

Background Daxx has been identified as a nuclear protein that involves in apoptosis and transcriptional repression. Daxx co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription. Human Daxx (hDaxx) is a protein that functions as a transcriptional regulation through its interaction with some DNA-associated proteins. The aim of this study was to explore the transcriptional regulatory effect of hDaxx interacting with adenovirus (Ad) 12 E1B (Ad12E1B) 55-kDa oncoprotein Methods The co-localization of hDaxx-Ad12E1B or hDaxx-PML protein in the nucleus was observed under a confocal microscope Interaction of hDaxx and Ad12E1B was analyzed by yeast two-hybrid assay Direct binding of hDaxx and Ad12E1B was analyzed using coimmunoprecipitation and Western blot in vivo and in vitro The activity of a luciferase reporter gene, which was regulated by an hDaxx modulated thymidine kinase (TK) promoter, was detected in an automat luminometer Results Ad12E1B, which co-localized with hDaxx in the nuclei of G401-CC3 cells, disrupted the co-localization of hDaxx and PML in the PML oncogenic domains (PODs) hDaxx bound directly to Ad12E1B in vivo and in vitro hDaxx interacted with Ad12E1B along its full length Ad12E1B enhanced transcriptional repression activity of hDaxx Conclusion Ad12E1B disrupts the co-localization of hDaxx with PML in PODs and enhances transcriptional repression activity of hDaxx

Four COVID-19 Cases of New Variant B.1.351 First Emerging in South Africa in Chinese Passengers on Same Flight-Shenzhen,China,January 2021

At 04∶50 on January 1,2021,a 36-year-old Chinese project manager(Case A),a 29-year-old Chinese worker(Case B),and a 53-year-old Chinese businessman(Case C)returned from Africa(Case A and B from South Africa and Case C from Lesotho)on the same flight and tested coronavirus disease 2019(COVID-19)RNA positive by real-time polymerase chain reaction(PCR)by Baoan District People’s Hospital.Shenzhen CDC received their oral nasopharyngeal swabs packages from the hospital and retested COVID-19 RNA positive at 09∶50.Meanwhile,Case D.