THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE / 药物分析学报

Objective To construct eukaryotic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humoral and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major capsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, E7Mxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry. After BALB/c mice were vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immunie adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal) , the great cellular immune responses were produced as revealed by delayed-type hypersensitivity (DTH) and lymphocyte proliferation, and the expression of IL-4 and IFN- γ cells in CD4+ and CD8+subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8+ IFN-γ + cells number, but CD4+ IL4+ cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immunoresponse. Conclusion These DNA vaccines produce remarkable cellular and humoral immuneresponses in the mouse and may provide as prophylatic and therapeutic candidates for HPV induced cancer treatment.

HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN CAN BE EXPRESSED IN LIVE ATTENUATED SHIGELLA FLEXNERI 5A STRAIN SH42 / 药物分析学报

Objective Attenuated strains of Shigella are attractive live vaccine candidates for eliciting mucosal immune responses which is a suitable carrier for the prophylactic human papillomaviruses (HPV) vaccine development, To examine the potential of a live Shigella based prophylactic HPV vaccine, HPV16L1should be expressed in attenuated shigella strain. Methods A Shigella large invasive plasmid (icsA/virG) based prokaryotic expression plasmid pHS3199 was constructed. HPV16L1 gene was inserted into plasmid pHS3199 to form pHS3199-HPV16 L1 construct, and pHS3199-hpv16L1 was electroporated into a live attenuated shigella strain sh42. The expression of HPV16L1 protein was demonstrated by Western blotting with monoclonal antibody to HPV16L1, The genetic stability of recombinant strain sh42-HPV16 L1 was monitored by consecutive passage culture. Invasive ability of sh42-HPV16L1 was evaluated by Hela cell infection assay. Results HPV16 L1 protein can be expressed in recombinant strain sh42-HPV16 L1, and the protein stably expressed over 140 generations. The invasive ability of sh42-HPV16L1 was diminished dramatically compared to its parent strain, but not abolished completely. Conclusion HPV16L1 protein was constitutively expressed in the attenuated strain of shigella flexneri sh42, and maintained partial invasive ability. Our strategy may represent a promising vaccine candidate against genital HPV16 infection.

Carboxyl terminus truncated human papillomavirus type 58 L1 protein maintains its bioactivity and ability to form virus-like particles / 华中科技大学学报（医学）（英德文版）

To prepare carboxyl terminus truncated human papillomavirus type 58 L1 (HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing--HPV58L1 protein were harvested and analyzed by SDS-PAGE and Western blot. The ProBond purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.

Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To develop an efficient expression, purification system of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization.</p><p><b>METHODS</b>A recombinant, pMMB68-LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60. The relevant target protein was identified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Sephacryl S-100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60. BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration. After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA.</p><p><b>RESULTS</b>rLTB protein was highly expressed in VSP60. After gel filtration with Sephacryl S-100, the purity of rLTB reached 98.1%, the yield rate was about 52%. After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL + rLTB groups were significantly increased, compared with the HEL alone group (P < 0.001).</p><p><b>CONCLUSIONS</b>A set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable. The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co-administered antigen.</p>

Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro.</p><p><b>METHODS</b>Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation.</p><p><b>RESULTS</b>Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.</p><p><b>CONCLUSIONS</b>Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.</p>

pcDNAL1 genetic immunization can induce specific cell-mediated immune responses in C57BL/6 mice / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the specific cell-mediated immune efficacy of the an HPV16 prophylactic vaccine.</p><p><b>METHODS</b>C57BL/6 mice were randomly divided into 3 groups: experimental group I (treated with pcDNA L1), control group II (treated with pcDNA3.1) and control group III (treated with PBS buffer). The mice were immunized three times during a three-week interval. Ten to fourteen days after the third inoculation, a footpad swelling test was used to detect delayed-type hypersensitivity (DTH) responses. Antigen-specific splenocyte proliferation assay and quantitation of IFN-gamma cells in splenocytes were performed by FACS assay.</p><p><b>RESULTS</b>In the experimental group, splenocytes actively proliferated after stimulation with HPV16 VLP, and had developed a markedly larger amount of CD8(+) IFN-gamma(+) cells, which is an index for special CTL. Also, the footpad was significantly thickened upon inoculation with HPV16 VLP.</p><p><b>CONCLUSION</b>Naked DNA vaccine of HPV16 L1 can induce specific cell-mediated immune responses in mice, which should be considered for evaluation of HPV16 DNA vaccine feasibility.</p>

IN VITRO CO-STIMULATORY ACTIVITY OF HUMAN B7.2(IgV+C)PROTEIN PRODUCED BY ENGINEERED BACTERIA / 药物分析学报

Objective To express human B7.2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-α. The fusion protein consisted of GST and hB7.2(IgV+C) was identified by SDS-PAGE and Western blotting.T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated by anti-CD3 antibody. Results The fusion protein GST-hB7.2 (IgV+C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7.2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

THE EUKARYOTIC EXPRESSION OF HUMAN PERFORIN PROTEIN AND THE ESTABLISHMENT OF HYBRIDOMAS PRODUCING ANTI-HUMAN PERFORIN PROTEIN / 药物分析学报

Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombinant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hybridoma cell lines secreting McAbs against human perforin were established, and the three McAbs showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as lgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.

CONSTRUCTION OF HUMAN INTERLUEKIN-18 DNA VACCINE AND IT'S EXPRESSION IN MAMMALIAN CELLS / 药物分析学报

Objective To construct the human interleukin-18(hIL-18) DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos-7 and D5.Methods Gene recombinant technique was used to construct hIL-18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL-18 eukaryotic expression vectors into Cos-7 and D5 cells.In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL-18 in Cos-7 and D5.Results The eukaryotic expression plasmid pVAX1-IL 18 was constructed successfully.hIL-18 was transiently expressed in Cos-7 and D5.Conclusion The eukaryotic expression plasmid pVAX1-IL 18 was constructed.In situ hybridization and Western Blot results proved the successful transient expression of pVAX1-IL 18 in Cos-7 and D5.Therefore,the work has settled the foundation for further biological research on hIL-18,including immunogene therapy through hIL-18.

The in situ expression of PDGF-B mRNA increased after the denudation of rabbit iliac arteries / 中国病理生理杂志

AIM: To elucidate the in vivo mechanisms of the proliferation of vascular smooth muscle cells (VSMCS) in injuried arteries. METHODS: A VSMCS proliferative model was constructed by injury of rabbit iliac arteries with balloon catheters and a probe designed for rabbit platelet-derived growth factor B chain (PDGF-B ) mRNA was used to detect the expression of it by intimal VSMCS on the vascular cross sections using an in situ hybridization technique at the indicated times. The relation of this expression to the proliferation of VSMCS by their expression of proliferating cell nuclear antigen (PCNA) and vascular intimal areas were estimated. RESULTS: The expression of PDGF-B mRNA of intimal VSMCS was increased when calculating the intimal PDGF-B mRNA positive cells per millimetre area at ?400 magnification with average numbers of 31.93?14.64 in 1 week group, 26 50?9 25 in 2 weeks group and 24 85?13 65 in 4 weeks group. This was in accordance with the expression of PCNA by VSMCS and the increase of intimal areas. CONCLUSION: The local production of PDGF-B by VSMCS via an autocrine mechanism is responsible for the continuous proliferation of these cells and formation of neointima after the injury. The probe designed is very useful for detecting rabbit PDGF-B mRNA.

ANALYSIS OF TYPE I AND TYPE Ⅱ CYTOKINES PROFILE OF LYMPHOCYTE IN PLEURAL EFFUSION OF NON-SMALL CELL LUNG CANCER PATIENTS / 药物分析学报

Objective To analyze mRNA expressions of 7 cytoklnes which influence the immune response in lym- phocytes in pleural effusion of non-small cell lung cancer patients to evaluate the effect of local tumor microenviron- ment on anti-tumor immune response and to explore the mechanism of tumor escape. Methods Detecting the mRNA expression of IL-2,INF-γ,IL-12,IL-18,IL-10,IL-4 and TGF-β 1 in lymphocytes in pleural effusion of non-small cell lung cancer patients and tuberculotic pleurisy patients on the single cell level by using in situ hybridization. Results In the pleural effusion of non-small cell lung cancer, the mRNA expressions of IL-10,TGF-β1 and IL-4 were signifi- cantly higher than those of IL-2,IL-12,IL-18 and INF-γ,as well as these of control group. The cytokine expression levels of tuberculotic pleurisy patients were very Iow, and there were no significant differences between different cy- tokines. Conclusion Type 2 cytokines are expressed predominantly in the pleural effusion of non-small cell lung can- cer. The increased co-expression of IL-10 and TGF-β1 indicates that they might act Jointly and play a critical role in the immunosuppression of non-small cell lung cancer.

Experimental Study on the Anti-tumor Mechanism of Interleukin-6 / 中国肿瘤生物治疗杂志

Segregated Kunming mice bearing S180 sarcoma were used as tumor models and treated with rhIL-6. The tumor regressed in most of the treated mice (8/10) in which there was no tumor growth when rechallenged with the same tumor cells, whereas 9/10 of controls died as the tumor progressed. Morphologically, in the treated group, the mice had enlarged spleen (4 times larger than that of control group)with hyperplastic white pulp consisted predominantly of activated lymphocytes. Cytotoxicity of spleen cells from IL-6 treated group to autologous tumor cells was higher than that of control group (907 ?318: 387 ?144, P=0.003) and so did L929 cell line used as target (1145?164: 186?251).We also set in vitro experiment using human PBL and human melanoma and colon carcinoma cell lines (A375,LS174). These cells were treated with IL-6 respectively and the cytotoxicity was assayed. Although PBL stimulated with IL-6 killed the LS174 more efficiently,the higher cytotoxicity to LS174 is because of the increased sensitivty of LS174 to the effector cells .On the contrary,IL-6 showed no effect on the A 375 cell line. It is assumed that this difference might result from the discrepancy of the recognition molecules existed on the cell surface between these two cell lines.

THE COMPARISION OF IN SITU HYBRIDIZATION BY USING ~3H WITH DIGOXIGENIN LABELED TNF PROBE / 西安交通大学学报(医学版)

The TNA mRNA expression in 33 samples of cervical carcinoma and 28 samples of condyloma have been observed by using ~3H and Digoxigenin labeled TNF—? cDNA probes and in situ hybridization technique, we found that the results of in situ hybrdfzation with these two kinds of labeled probes were alike, the sensitivity with ~3H labeled probe was slightly higher than that with Digoxigenin labeled probe. Nevertheless, the nonisotope probe would be used more and more in future because of its safety, rapidity and convenience in work.

USING WEHI CELL LINE AS A TARGET AND MTT COLORIMETRIC TECHNIQUE FOR MONOCYTES CYTOTOXIC ASSAY / 西安交通大学学报(医学版)

We used WEHI clone—13 cell line as target and MTT colorimetric technique to evaluate monocyte cytotoxicity, this method is superior to the traditional~(51) Cr release technique. It is more sensitive,more rapid and less effectors needed, as well as no isotope contamination, and it is mose suitable in clinic.

THE EFFECT OF POLYACTIA B ON TUMOR-INFILTRATING IMMUNOCOMPETENT CELLS WITIHN MURINE S_(180) SARCOMA / 西安交通大学学报(医学版)

Polyactin B (Pb) has been proved to have the effect of considerable tumor suppression. Recently,we used murine S_(180) Sarcoma as a model and observed the effect of Pb on the tumor—infiltrating immunocompetent cells, and also the infiltrating neutrophils and the alteration of small blood vessels within the tumor tissue. Compared with control: in Pb—treated group, there were more L_3T_4~+and Lyt_2~+ lymphocytes infiltrating in the periphery of the tumor ,and also within the tumor. In addition, the tumors had more prominant hyperemia, micro—thrombosis and neutrophil infiltration around the necrotic areas. The present findings suggest that the tumor—suppression effect of Pb might be mediated through TNF produced by immunocompetent cells.