鸡柔嫩艾美球虫子孢子表面抗原基因λMzp5-7在Hela细胞中的表达

将已构建的柔嫩艾美球虫(Eimeria tenella)子孢子表面抗原pMD-Mzp5-7基因片段定向亚克隆到核酸疫苗载体pVAX1上,构建了真核表达载体pVAX-Mzp5-7,酶切鉴定正确后,用脂质体介导的转染法转染Hela细胞,进行了体外表达;并用裸DNA质粒经肌肉接种鸡体内表达。经Western-blotting及RT-PCR鉴定,表明该基因在Hela细胞及鸡体内均获得表达,并有一定的反应原性。

双启动子shRNA表达载体对hTERT和bcl-2基因表达的抑制作用

目的:研究双启动子shRNA表达载体对人端粒酶蛋白催化亚单位(hTERT)和B细胞淋巴瘤/白血病-2(bcl-2)基因表达的抑制作用。方法:分别从构建好的大肠杆菌DH5α菌体中提取含有双启动子shRNA的表达载体pdPRO-TB、单启动子对照pdPRO-T、pdPRO-B和阴性对照质粒pdPRO,通过脂质体介导瞬时转染Hela细胞,倒置显微镜下观察细胞形态。实时荧光定量RCR检测hTERT和bcl-2基因的表达水平。结果:4种菌体中均提取出5 000 bp左右的质粒,分别转染Hela细胞48 h后,可见转染pdPRO的Hela细胞形态无明显变化,转染pdPRO-T和pdPRO-B的Hela细胞有部分悬浮,转染pdPRO-TB的Hela细胞有大量悬浮。各载体质粒构建均成功,各目的基因相对管家基因的含量:T(hTERT)=39.8%,B(hTERT)=107.9%,TB(hTERT)=32.1%;T(bcl-2)=91.4%,B(bcl-2)=35.4%,TB(bcl-2)=31.4%。结论:双启动子shRNA表达载体构建成功,可同时抑制hTERT和bcl-2基因的表达。

MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION

To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned based on RT PCR amplification from HeLa cells and sequenced The cloned hTRTcDNA was in frame inserted into His tag fusion expression vector pEK318 The His tag hTRT fusion proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI and BamHI sites DNA sequencing showed the isolated fragment was consistent to those reported SDS PAGE present that a 17kDa protein was expressed stably in E coli JM109 harboring pEKTRT344 containing 6×His tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6×His tag and hTRT 243aa was only detectable as 27 kDa band in western blotting Both of fusion proteins were purified by Ni NTA chromatography and showed single band(>95% purifity) in Coomassie Brilliant staining Western blotting confirmed that two proteins could be recognized by the Ni NTA AP conjugate Conclusions. The hTRT catalytic domain was highly conserved The expressed hTRT protein contained recognizable His tag, telomerase specific and strong antigenic epitops, which may be convenient for further investigation

The proliferation inhibition of Hela cells by histone deacetylases- 1 siRNA

Objective： To investigate the anti-proliferative effect of histone deacetylases-1 （HDAC-1） knockdown in Hela cells. Methods： The HDAC-1 protein was knockdowned using siRNA. The expression of HDAC-1 was detected by Western blotting. Apoptosis was assessed by flow cytometry. The inhibition of cell growth was assesses by MTT assay. Results： HDAC-1 siRNA knockdowned the expression of HDAC-1 protein. HDAC siRNA inhibited the proliferation of Hela cells. HDAC- 1 siRNA induced apoptosis. Conclusion： HDAC-1 siRNA may inhibit the growth of Hela cells by inducing apoptosis.

Effect of aspirin alone or combined with cisplatin on human cervical carcinoma HeLa cells

Objective： To study the effect and mechanism of aspirin alone or combined with cisplatin （DDP） on human cervical carcinoma HeLa cells. Methods： HeLa cells were treated by different concentrations of aspirin, DDP alone or both. The inhibitory effect on cell growth was analyzed by MTT and colony-forming assay. Cell apoptosis was measured with flow cytometry. The mRNA levels ofBcl-2, Bax and NF-κB（P65） were studied by RT-PCR. Results： MTT assay showed that aspirin inhibited HeLa cell proliferation in a time-and dose-dependant maoner. Aspirin decreased clone numbers in colony formation assay. Aspirin also induced apoptosis of HeLa cells in a dose- and time-dependent manner as detected by flow cytometry. The inhibition effects on proliferation, colony formation and apoptosis were significantly enhanced when cells were treated with both aspirin and DDP. RT-PCR demonstrated that aspirin decreased the transcription of Bcl-2 and NF-κB, and increased expression of Bax gene. Conclusion： Aspirin can induce apoptosis in HeLa cells. Combination of aspirin and DDP displays a synergistic effect. The possible mechanism might be that aspirin downregulates the mRNA levels of Bcl-2 and NF-κB gene and upregulates the expression of Bax.

Construction and expression of the bicistronic expression vector with RANTES and SDF-1 genes

Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.

Inhibition of SIRT1 Increases EZH2 Protein Level and Enhances the Repression of EZH2 on Target Gene Expression

Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were established by in-fection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells. Results Western blot results showed that EZH2 protein level increased upon SIRT1 de-pletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter region of EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases. Conclusions Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expres-sion.