[Construction and expression characteristics in vitro of human papillomavirus type 16 E7 suicidal DNA vaccine].

OBJECTIVE: To construct the suicidal DNA vaccine of human papillomavirus type 16 E7 gene (HPV16), and explore the DNA vaccine expression characteristics in vitro and capacity of inducing the transfected cells into apoptosis. METHODS: HPV16 E7 gene cloned by PCR from pET32/E7 was inserted into the plasmid pSCA1 to construct the recombinant plasmid pSCA/E7, followed by identification with PCR, BamH I and Sma I digestion and sequencing. pSCA/E7 was then used to transfect BHK-21 cell line. The transient expression of HPV16 E7 gene was confirmed by immuno-fluorescent staining, and the apoptosis induced by pSCA/E7 was checked with TDT-mediated dUTP nick end-labeling (TUNEL). RESULTS: The cloned E7 gene fragment was about 400 bp in length. PCR, restriction endonuclease digestion and sequence analysis revealed that the HPV16 E7 gene was cloned into the eukaryotic expression plasmid pSCA1 successfully. Immunofluorescent staining confirmed that the E7 gene could express in BHK-21 cell line. The BHK-21 cells transfected with pSCA/E7 could be induced into apoptosis which was confirmed by TUNEL. CONCLUSION: The results show that HPV16 E7 suicidal DNA vaccine can express in BHK-21 cell line, and induce the pSCA/E7 transfected cells into apoptosis. These findings may provide the foundation for exploring the therapeutic vaccine against HPV16-associated cervical cancer.

[Steady expression and activity determination of recombinant signal peptide of mBD2 in SiHa cell].

OBJECTIVE: To observe the expression pattern and effect of recombinant murine beta defensin 2 (rmBD2) on the proliferation of cell transfected with pcDNA3. 1 (+)/rmBD2. METHODS: The recombinant plasmid pcDNA3. 1 (+)/rmBD2 was transferred into SiHa cells. The transfected SiHa cells were selected by G418 with 100 microg/mL for over 20 days. The steady expressions of rmBD2 protein and rmBD2 mRNA were detected by immunofluorescence and RT-PCR. The effects of rmBD2 on SiHa cell growth and reproduction were measured by MTT. RESULTS: The SiHa cells which could stably express the rmBD2 protein were harvested with 100 microg/mL of G418. The rmBD2 protein expression was, by immunofluorescence, confirmed and its mRNA in SiHa with rmBD2 could be detected at 4 weeks, 6 weeks, 8 weeks, and 10 weeks after cell transfected. A 220 bp segment encoding rmBD2 was amplified by RT-PCR and proved by sequencing. The SiHa/K cells transfected pcDNA3. 1 (+) and SiHa had no expression of rmBD2 protein. The SiHa with rmBD2 expression grew slowly than SiHa/K and SiHa control (P < 0.05). CONCLUSION: The screening for SiHa with rmBD2 expressing stably is successful. The cell growth curve shows that rmBD2 can inhibit the proliferation of tumor cells. The above results establish a solid foundation for further studying the biological properties and the anti-tumor mechanism of beta defensins.

[Cloning and eukaryotic expression of murine beta-defensin-2(mBD-2)].

AIM: To clone murine beta defensin-2 gene (mBD2) and to express the mBD2 protein eukaryotically. METHODS: Total RNA was isolated from the lungs of BALB/c mice which were injected with LPS in advance. The DNA fragment encoding mBD2 was amplified by RT-PCR and inserted into the plasmid pcDNA3.1(+), which was then digested with EcoR I and Xho I to construct the recombinant plasmid, pcDNA3.1(+)/mBD2. The pcDNA3.1(+)/mBD2 was identified by endonuclease digestion, PCR, and sequencing analysis. The SiHa cells were transfected with pcDNA3.1(+)/mBD2 plasmid and screened by G418 of 100 mg/L over 20 days. Steady expression of mBD2 was confirmed by immunofluorescent staining and RT-PCR. RESULTS: About 250 bp DNA fragment was amplified by RT-PCR from lung total RNA of the mice injected with LPS. The eukaryotic expression vector, pcDNA3.1(+)/mBD2, was successfully constructed after inserting the mBD2 fragment into pcDNA3.1(+). Most of SiHa cells transfected with pcDNA3.1(+)/mBD2 and screened by G418 could express the mBD2 protein, confirmed by immunofluorescent staining and RT-PCR. CONCLUSION: The eukaryotic vector of pcDNA3.1(+)/mBD2 was successfully constructed and transfected into SiHa cells, which established a solid foundation for further study on the biological characteristics and anti-tumor mechanisms of the mBD2 protein.

[Cloning of ureI gene from Helicobacter pylori and its expression in E. coli].

OBJECTIVE: To clone the urea membrane channel gene (ureI) from Helicobacter pylori (Hp) for its expression in E. coli, and evaluate the expression conditions and immunological features of the fusion protein. METHODS: ureI gene cloned by PCR from Hp was inserted into the plasmid pET32a (+) to construct the recombinant plasmid pET32a/ureI, followed by identification by BglII and HindIII digestion and sequencing. E. coli BL-21+(DE3) was transformed with pET32a/ureI to obtain the engineered bacterium BL21+/UreI, which was cultured at different temperatures and induced with 1.0 mmol/L IPTG for expression of the recombinant protein. The expressed proteins were identified by SDS-PAGE and analyzed by Pro-gel analyzer 4.0. Western blotting was performed to evaluate the immunogenicity of the expressed protein. RESULTS: The cloned gene fragment was about 650 bp in length, and BglII and HindIII digestion of pET32a/ureI yielded a 650-bp band. Sequence analysis revealed that the cloned ureI gene contained 646 bp without reading frame alterations. Comparison against GenBank indicated a homology of 100% of the cloned gene with ureI gene of the corresponding Hp strains, and also one no less than 98.5% with ureI gene from other strains. The engineered E. coli BL21+/UreI could express recombinant UreI (rUreI) with His tag, and the target protein accounted for 20.2% of the total bacterial proteins after 1.0 mmol/L IPTG induction of the bacterium at 37 degrees C for 14 h. SDS-PAGE and Western blotting showed that the recombinant UreI protein was produced mainly in the inclusion bodies and fused with his-tag (rUreI/his), which could react with human anti-Hp and mAb to his tag but not with mAb to Hp UreB. CONCLUSIONS: We have successfully cloned ureI gene and constructed the prokaryotic expression plasmid for efficient rUreI expression, and the fusion protein rUreI/his expressed in the inclusion bodies can react specifically with both Hp antibody and his-tag antibody.

[Analysis of protein expression feature and construction of procaryotic expression system for human papillomavirus type 16 (HPV-16) E4 gene].

OBJECTIVE: To clone the E4 gene (E4) from human papillomavirus type 16(HPV-16), construct the engineering bacteria of prokaryotic expression, and explore the expression conditions and the characters of expression product. METHODS: The complete E4 gene was cloned by PCR from the sample cell extract of clinical cervical disease that was the positive HPV-16 confirmed by Real-PCR. The E4 DNA fragment was inserted into the pET32a(+) to construct a prokaryotic expression plasmid, called as pET32/E4. Then the expression plasmids were transferred into competent E. coli BL21 (DE3). Recombinant DNA was identified by Bgl II and Hind III digestion, and then sequencing. The recombine bacterium, BL21/E4, was induced with different IPTG concentrations at different temperatures. The expressed proteins were checked and analyzed by SDS-PAGE and Gel-Pro Analyzer 4. His-tag of BL21/E4 expression protein was hybridized to McAb. RESULTS: The E4 gene cloned by PCR was about 342 bp. The blasted result showed that the E4 gene had 99% homology of HVP-16 DNA sequence, the cloned E4 gene expression frame was the same as HVP-16 East Asia strain's. Compared with other HPV-16 strains in GenBank, the homology of E4 gene was above 97%. pET32/E4 could express recombinant E4 (rE4) in BL21. The highest expression, which was 12.2% or 12.8% of total bacterial proteins respectively, was gotten when BL21/E4 was induced by 0.1 mmol/L IPTG at 28 degrees C or 37 degrees C for 18 hours. The results of SDS-PAGE and Western blot showed the rE4 was expressed mainly to form the inclusion body, and to fuse with his-tag (rE4/His), that was soluble and had a molecular weight as about 34 KDa. CONCLUSION: We cloned successfully the E4 gene from HPV-16 and constructed the prokaryotic expression E. coli BL21/E4, which could expression rE4 protein fused with his-tag (rE4/His), effectively. The fused protein could react to McAb recognizing His-tag, which was convenience purified by affinity chromatography. The above research results built a good foundation for preparing the high grade of purity E4 protein and developing the relative study.

[Construction of eukaryotic expressing plasmids for HA and HA1 of influenza A virus and their transient expression in HEK293 cells].

OBJECTIVE: To construct influenza A virus (A/PR/8/34) HA and HA1 eukaryotic expressing plasmids and study their expression in HEK293 cells. METHODS: HA and HA1 genes were cloned by RT-PCR and then inserted into pcDNA3. 1 (+). After identification of restriction enzyme digestion, PCR and sequencing analysis, HA and HA1 eukaryotic expressing plasmids were transfected into HEK293 cells with PolyFect Transfection Reagent. Immunofluorescence assay was used to observe the transient expressing result. RESULTS: It was confirmed that the construction of HA and HA1 eukaryotic expressing plasmids was made successfully. The stronger fluorescence signals were detected in transfected HEK293 cells with these two kinds of plasmids by immunofluorescence assay. CONCLUSION: The experiment is a success in the construction of eukaryotic expressing plasmids for HA and HA1, thus providing a basis for further probing into the mechanism of virus infection and exploring DNA vaccine.

[Prokaryotic expression and identification of human papillomavirus type 16 E7 protein encoding gene].

OBJECTIVE: To construct the prokaryotic expression plasmid pET32/E7 and express the human papillomavirus type 16 E7 protein in E. coli. METHODS: HPV16 E7 gene was amplified by PCR. The amplified E7 fragment was inserted into the plasmid pET32a (+) that was digested with BamH I and Hind III. The recombinant plasmid pET32/E7 was transformed into E. coli JM109 which was selected with ampicillin. The positive clones containing recombinant plasmid pET32/E7 were verified by BamH I and Xho I digestion, and then sequenced. HPV16 E7-TRX recombinant protein expression in the E. coli BL21(ED3) was identified by SDS-PAGE and Western blot. RESULTS: The prokaryotic recombinant plasmid pET32/E7 was successfully constructed. The BL21(DE3) transformed recombinant plasmid pET32/E7 had expressed HPV16 E7-TRX recombinant protein effectively. Under the conditions of 1 mmol/L IPTG and 30 degrees C, the amount of HPV16 E7-TRX recombinant protein was about 30% of bacterial total proteins. CONCLUSION: The construction of the prokaryotic recombinant plasmid pET32/E7 and the successful expression of the recombinant protein HPV16 E7-TRX would strongly promote the research of the biological properties and the transformational mechanism of the HPV16 E7 protein on the specific cells.

[Expression and immunogenicity analysis of a recombinant fusion protein of V. Cholera ctB and H. pylori ure I].

AIM: To express fusion protein of the cholera toxin B subunit (ctB) and the urea membrane channel gene (ure I) of H. pylori in E. coli, and analyze its immunogenicity. METHODS: The prokaryotic expression vector pET32a+/ctB/ure I was constructed by inserting ctB gene amplified by PCR into the 5' terminus of ure I gene of expression vector pET32a+/ure I. The fusion gene was verified by endonuclease digestion and sequence analysis. The fusion protein ctB/ure I was expressed in E. coli BL21(DE3), purified by His-HP affinity chromatography, and analyzed by SDS-PAGE, Western blot and Pro-gel analyzer 4.0. The mice were immunized with purified ctB/ure I, and the immunoreactivity with ctB and ure I of the murine sera was analyzed by indirect ELISA. RESULTS: The pET32a+/ctB/ure I expression vector was constructed successfully and confirmed by endonuclease digestion and sequence analysis. The expressed ctB/ure I protein with molecular weight about 58,000 was shown when induced with 1 mmol/L IPTG for 4 h at 22 degrees C, and the protein could react with horse anti-ctB and human anti-ure I sera when detected with Western blot, and the purity of the purified protein was about 94.3%. The sera from mice immunized with purified ctB/ure I protein could react with ctB, ure I, and ctB/ure I when detected with indirect ELISA. CONCLUSION: The fusion protein expression vector pET32a+/ctB/ure I was constructed successfully. The fusion protein ctB/ure I was shown to have immunoreactivity with both anti-ctB and anti-ure I anti-sera, and could evoke production of anti-ctB and anti-ure I antibody in mice. Our work established a good foundation for further study on the new and effective H. pylori vaccines.

[Prokaryotic expression and identification of human papillomavirus type 16 E5 protein].

OBJECTIVE: To construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH(3)T(3) cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5). METHODS: HPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plasmid pET32a(+) followed by digestion with BamH I and Hind III. The recombinant plasmid pET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BamH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E. coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH(3)T(3) cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR. RESULTS: The pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 degrees C, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfected NIH(3)T(3) cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment. CONCLUSIONS: The prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E .coli and NIH(3)T(3) cells with stable expression of HPV16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.