[The influences of Tet system on activity and tissue specificity of HBV Cp.].

AIM: Analyze the effect of Tetracycline-controlled system on Hepatitis B virus core promoter (Cp) activity and tissue specificity. METHODS: The 7 Teto sequence was amplified from plasmid pTL-8 using polymerase chain reaction (PCR) and cloned into T vector pMD19-T Simple. After sequencing, the 7 teto sequence was subcloned into upstream of Cp in pGL3-Basic/Cp. In order to observe the effect of Tetracycline-controlled system on Cp activity and tissue specificity, the plasmid pTL-8 which luciferase encoding sequence was excised and pGL3-Basic/7t/Cp were cotransfected into different tissue-derived cell lines including HepG2, Hela, COS-7, MDA-MB-231 and HT-29. The dual luciferase activities were determined by Dual Luciferase Report (DLR) assay system. RESULTS: Have successfully constructed plasmid pGL3-Basic/7t/Cp. The transcriptional activity of Cp was increased significantly after the teto sequence was cloned upstream of Cp. CONCLUSION: The transcriptional activity of Cp is augmented significantly by Tetracycline-controlled system. But the tissue specificity of Cp lost at the same time.

[DNA/MVA combined immunization: antibody response to Plasmodium falciparum merozoite surface protein 1 in mice].

OBJECTIVE: To explore the effect of DNA/MVA combined immunization in enhancing antibody response to MSP1. METHODS: DNA vaccine and recombined MVA were constructed based on synthesized MSP1 gene (3D7). BALB/c mice were primed with DNA solely or together with GM-CSF expressing plasmid and then boosted with rMVA/ 190. Serum IgG and subtype IgG1 and IgG2a were assayed by ELISA. All mice were challenged with allelic replaced Plasmodium berghei. RESULTS: Antibodies to MSP1-190 were detected after DNA immunization with an end-point dilution titer of 1:2500. When GM-CSF plasmid was added, the antibody end-point dilution titer reached 1:11150, with an increase of 53 and 10 times respectively after MVA boosting. Among them anti-19000 antibodies were prominent, 1/4-1/3 of total IgG in serum. However, when the mice were challenged with Pb-PfM19 no prolonged survival was observed (P>0.05). CONCLUSION: High titer antibodies can be elicited in mice by using codon optimized MSPI gene and DNA/MVA combined immunization. The specificity and protection of these antibodies is being further investigated.

[Modulation of immune response to Plasmodium falciparum apical membrane antigen 1 DNA vaccine by cytokine plasmids].

OBJECTIVE: To explore the effect of cytokine encoding plasmids on DNA immunization in mice. METHODS: Prototype DNA vaccine VR1020/E which contain Plasmodium falciparum apical membrane antigen 1 (AMA1) ectodomain was constructed, and eukaryotic expression vectors pcDNA3/GM-CSF, pcDNA3.1(-)/IL-4, pIL-12 and pGM-CSF/pTPA-E were also built. BALB/c mice were immunized with VR1020/E alone or with VR1020/E plus different cytokine plasmids. Serum IgG and its subtype were determined by ELISA and in vitro splenocyte proliferation assay was done. RESULTS: GM-CSF, IL-4 and IL-12 encoding plasmids all promoted mice immune response to VR1020/E, the antibody level increased 7 to 10 times and splenocyte proliferation was enhanced too. Plasmid pcDNA3/GM-CSF induced much more IgG1 whereas plasmid pIL-12 induced much more IgG2a. CONCLUSION: Cytokine encoding plasmids might be used as adjuvant in AMA1 DNA immunization.

Effects of tetracycline operator element insertion on Plasmodium falciparum glycophorin binding protein 130 gene promoter activity.

OBJECTIVE: To construct tetracycline operator (TetO) modified glycophorin binding protein 130 gene (GBP130) promoter of Plasmodium falciparum and investigate the position effect of insertion of TetO on the promoter activity. METHODS: Cloning of 7-copy of TetO (7cot) sequence into 4 points relative to transcriptional initiation site of GBP130 promoter in pGBPCATdelta2 plasmid (2 upstream and 2 downstream), respectively, produced 4 derivative plasmids, pG/7T (-5), pG/7T (-2), pG/7T (+2) and pG/7T (+5). After transient transfection, the expression level of reporter gene CAT in both pGBPCATdelta2 and its derivative plasmids was detected and analysed by CAT ELISA. RESULTS: Identification by enzyme restrictions and PCR amplifications, as well as DNA sequencing confirmed that the plasmids were successfully constructed. Transfection of these plasmids and CAT detection suggested that insertion of 7cot into each point of GBP130 promoter enhanced the promoter activity, and downstream location of 7cot in the promoter showed higher promoter activity than those located upstream, with the strongest effect in Ins 4 (point +5). CONCLUSION: Plasmid pG/7T (+5), in which 7cot was inserted into +5 point of GBP130 promoter, can be chosen as the responsive plasmid in establishing tetracycline-controlled transgenic expression system of malarial parasites.

[Preliminary study on regulable DNA vaccines against Plasmodium falciparum].

AIM: To construct regulable DNA vaccine against Plasmodium falciparum by using tetracycline(Tet) regulable system. METHODS: Eukaryotic expression vectors pTL-8/apical membrane antigen 1 (AMA-1) (tTA) and pTL-8/AMA-1(rtTA) gene which express trans-activator (tTA) or reverse trans-activator(rtTA), respectively, and AMA-1 gene of Plasmodium falciparum were constructed. BALB/c mice were immunized with these plasmids and doxycycline (dox) was administered to regulate the expression of AMA-1. For some mice immunized with pTL-8/AMA-1(rtTA), pUHS6-1, a plasmid containing trans-silencer (tTS) to suppress basal expression of AMA-1 from pTL-8/AMA-1(rtTA), was injected into these mice together with pTL-8/AMA-1(rtTA). The sera of the mice were isolated at 2,4,6 and 8 weeks post-immunization and the antibodies specific to AMA-1 were measured by ELISA. RESULTS: pTL-8/AMA-1 and pTL-8/AMA-1(rtTA) were constructed successfully. The mice immunized by pTL-8/AMA-1(tTA) with dox or by pTL-8/AMA-1(rtTA) without dox (at these conditions, AMA-1 was expressed at basal level)developed significant antibodies against AMA-1. Mice immunized by pTL-8/AMA-1(rtTA) and pUHS6-1 without dox did not develop significantly antibodies against AMA-1. In contrast, the mice immunized by pTL-8/AMA-1(rtTA) and pUHS6-1 with dox produced high level of antibodies. CONCLUSION: pTL-8/AMA-1(rtTA) combined with pUHS6-1 is a good regulable DNA vaccine candidate against Plasmodium falciparum.

[Protective antibody response to recombinant fragments of Plasmodium falciparum apical membrane antigen 1].

OBJECTIVE: To determine the role of putative apical membrane antigen (AMA)1 domains in inducing protective immunity and to provide basis for selection of vaccine applicable segments. METHODS: Encoding gene segments of AMA1 were amplified and cloned into pET prokaryotic expression vectors. Recombinant proteins were expressed and purified. Groups of BALB/c mice were immunized by using recombinant protein in Freund's adjuvant, and the IgG titer and specificity of the immune sera were analyzed by IFA and Western blotting. Efficiency of the immune sera in inhibiting Plasmodium falciparum in vitro growth was evaluated. RESULTS: Recombinant AMA1 fragments including the entire ectodomain E and subdomain I + II, I, II and III were successfully expressed and purified. Different levels of antibody were induced in mice by individual proteins and all the immune sera recognized native antigen in the parasites. Sera from protein E and I + II immunized mice inhibited the growth of parasites. CONCLUSION: The integrality of the ectodomain of AMA1 determines the conformation of the protective antibody epitopes, and these protective epitopes distribute mainly in the subdomain I.