Regulation of Disease-Resistance Genes against CWMV Infection by NbHAG1-Mediated H3K36ac.

Post-translational modification of proteins plays a critical role in plant-pathogen interactions. Here, we demonstrate in Nicotiana benthamiana that knockout of NbHAG1 promotes Chinese wheat mosaic virus (CWMV) infection, whereas NbHAG1 overexpression inhibits infection. Transcriptome sequencing indicated that a series of disease resistance-related genes were up-regulated after overexpression of NbHAG1. In addition, cleavage under targets and tagmentation (Cut&Tag)-qPCR results demonstrated that NbHAG1 may activate the transcription of its downstream disease-resistance genes by facilitating the acetylation level of H3K36ac. Therefore, we suggest that NbHAG1 is an important positive regulator of resistance to CWMV infestation.

A papain-like cysteine protease-released small signal peptide confers wheat resistance to wheat yellow mosaic virus.

Wheat yellow mosaic virus (WYMV), a soil-borne pathogen, poses a serious threat to global wheat production. Here, we identify a WYMV resistance gene, TaRD21A, that belongs to the papain-like cysteine protease family. Through genetic manipulation of TaRD21A expression, we establish its positive role in the regulation of wheat to WYMV resistance. Furthermore, our investigation shows that the TaRD21A-mediated plant antiviral response relies on the release of a small peptide catalyzed by TaRD21A protease activity. To counteract wheat resistance, WYMV-encoded nuclear inclusion protease-a (NIa) suppress TaRD21A activity to promote virus infection. In resistant cultivars, a natural variant of TaRD21A features a glycine-to-threonine substitution and this substitution enables the phosphorylation of threonine, thereby weakening the interaction between NIa and TaRD21A, reinforcing wheat resistance against WYMV. Our study not only unveils a WYMV resistance gene but also offers insights into the intricate mechanisms underpinning resistance against WYMV.

First-line induction chemotherapy with high-dose methotrexate versus teniposide in patients with newly diagnosed primary central nervous system lymphoma: a retrospective, multicenter cohort study.

BACKGROUND: This study aimed to compare the efficacy and safety of high-dose methotrexate (HD-MTX) versus teniposide (TEN) in patients with newly diagnosed immunocompetent primary central nervous system lymphomas (PCNSLs). METHODS: The study included immunocompetent, adult patients with newly diagnosed PCNSL at 22 centers in China from 2007 to 2016. The patients received HD-MTX or TEN as first-line induction therapy. The objective response rate, progression-free survival, and overall survival were analyzed for each patient cohort. RESULTS: A total of 96 patients were eligible: 62 received HD-MTX, while 34 received teniposide. The overall response rate was 73.2% and 72.7% in the MTX and the TEN cohorts, respectively (P = 0.627). The median progression-free survival was 28.4 months [95% confidence interval (CI): 13.7-51.2] in the MTX cohort and 24.3 months (95% CI: 16.6-32.1) in the TEN cohort (P = 0.75). The median overall survival was 31 months (95% CI: 26.8-35.2) in the MTX cohort and 32 months (95% CI: 27.6-36.4) in the TEN cohort (P = 0.77). The incidence of any grade of coagulopathy/deep-vein thrombosis and gastrointestinal disorders was significantly higher in the MTX cohort than in the TEN cohort; no significant difference was found in the incidence of other adverse events between the two cohorts. CONCLUSIONS: This was the first multicenter study using TEN as the main agent compared with HD-MTX in newly diagnosed primary CNS lymphoma. The TEN-based regimen was non-inferior to the HD-MTX-based regimen with similar overall responses. CLASSIFICATION OF EVIDENCE: This study provided Class III evidence that the teniposide-based regimen was non-inferior to high-dose methotrexate - based regimen with similar overall responses and long-time survival in immunocompetent patients with PCNSL.

Hypomethylation and the Resultant Overexpressed PARM1: a Biomarker for Poor Prognosis of Diffuse Large B-cell Lymphoma.

Prostate androgen-regulated mucin-like protein (PARM1) is known to promote cell survival via protecting the cell surface, thus being involved in cancer development. The Gene Expression Profiling Interactive Analysis (GEPIA), MEXPRESS database, LinkedOmics database, GeneMANIA database, and the Tumor Immune Estimation Resource (TIMER) database were accessed to explore the epigenetic regulation, prognostic value, biological functions and mechanisms of PARM1 in diffuse large B-cell lymphoma (DLBCL). Hypomethylation and resultant overexpression of PARM1 was found in DLBCL. The high-level expression of PARM1 was related to the poor outcome of DLBCL patients. PARM1 participated in DNA repair, cell cycle, and cellular response to stress. PARM1 was also associated with autophagy, apoptosis, Ras pathway, and MAPK cascade. Significant kinase targets of PARM1 included ATM, CDK1, and CDK2. Significant transcription factor targets of PARM1 involved ELK1, MYC and so on. Significant miRNA targets of PARM1 included miR21, miR202, miR323, and miR345. Further analysis suggested that the PARM1 regulated autophagy through the PI3K-Akt signaling. PARM1 was found to be correlated with immune cell infiltration, which indicated the important roles of PARM1 in microenvironment of DLBCL. Our study lays a foundation for further research on the impact of PARM1 in DLBCL tumorigenesis and precision therapy.

[Clinical Characteristics and Long-term Prognosis Analysis of Patients with Primary Bone Lymphoma].

OBJECTIVE: To analyze the clinical characteristics and long-term prognosis of patients with primary bone lymphoma (PBL). METHODS: The clinical data of 21 patients with PBL treated in our center from 2005 to 2018 were analyzed retrospectively, the clinical characteristics and the factors affecting prognosis of the patients were analyzed. RESULTS: The median age of all the 21 newly diagnosed PBL patients was 40(12-71) years old. Ostealgia was the initial symptom in most of the patients (19/21,90.5%). 42.9%(9/21) of the patients showed single bone lesion only. 571% (12/21) of the patients showed diffuse large B cell lymphoma. 28.6% (6/21) of the patients showed anaplastic large cell lymphoma and 9.5% (2/21) of the patients showed T cell lymphoblastic lymphoma. All the patients received chemotherapy (CHOP or CHOP like regimen, 33.3% plus rituximab) with or without radiotherapy and/or autologous hematopoietic stem cell transplantation (ASCT). 18 patients achieved clinical remission (including 15 for CR and 3 for PR). The median follow-up time was 48 months. The 5-year overall survival rate and progression-free survival rate of the patients were was 67.5% and 63.7%, respectively. The single factors analysis showed that ASCT was the important prognostic factor of PFS, while the single or multiple bone lesion was the factors affecting OS of the patients. There were no statistical differences with the effects of age, sex, stage, ECOG score, LDH level, B symptoms and radiotherapy for the prognosis of patients. CONCLUSION: Diffuse large B cell lymphoma is the most common pathological type of PBL. Chemotherapy is the main treatment, which can be combined with radiotherapy and/or ASCT. The ASCT and the number of bone lesion are the factors for long time survival of the patients.

Genome-Wide Identification and Characterization of the Cystatin Gene Family in Bread Wheat (Triticum aestivum L.).

Cystatins, as reversible inhibitors of papain-like and legumain proteases, have been identified in several plant species. Although the cystatin family plays crucial roles in plant development and defense responses to various stresses, this family in wheat (Triticum aestivum L.) is still poorly understood. In this study, 55 wheat cystatins (TaCystatins) were identified. All TaCystatins were divided into three groups and both the conserved gene structures and peptide motifs were relatively conserved within each group. Homoeolog analysis suggested that both homoeolog retention percentage and gene duplications contributed to the abundance of the TaCystatin family. Analysis of duplication events confirmed that segmental duplications played an important role in the duplication patterns. The results of codon usage pattern analysis showed that TaCystatins had evident codon usage bias, which was mainly affected by mutation pressure. TaCystatins may be regulated by cis-acting elements, especially abscisic acid and methyl jasmonate responsive elements. In addition, the expression of all selected TaCystatins was significantly changed following viral infection and cold stress, suggesting potential roles in response to biotic and abiotic challenges. Overall, our work provides new insights into TaCystatins during wheat evolution and will help further research to decipher the roles of TaCystatins under diverse stress conditions.

Methylation silencing CDH23 is a poor prognostic marker in diffuse large B-cell lymphoma.

Cadherin-23(CDH23) mediates homotypic and heterotypic cell-cell adhesions in cancer cells. However, the epigenetic regulation, the biological functions, the mechanisms and the prognostic value of CDH23 in diffuse large B-cell lymphoma (DLBCL) are still unclear. The Gene Expression Profiling Interactive Analysis (GEPIA) and the Gene Expression Omnibus (GEO) database were employed to analyze the CDH23 expression level in DLBCL. The correlation of CDH23 expression and methylation was analyzed by LinkedOmics database. The prognostic value was analyzed via GEPIA. Correlated genes, target kinase, target miRNA, target transcription factor and biological functions were identified by LinkedOmics and GeneMANIA database. The relationship between CDH23 and the immune cell infiltration was explored by the Tumor Immune Estimation Resource (TIMER). The expression of CDH23 was reduced by DNA methylation significantly in DLBCL tissue. Reduction of CDH23 represented poor outcome of DLBCL patients. Functional enrichment analysis showed that CDH23 mainly enriched in cancer cell growth, cell metastasis, cell adhesion, cell cycle, drug catabolic process, leukocyte mediated immunity and DNA repair by some cancer related kinases, miRNAs and transcription factors. These results indicated that methylated reduction of CDH23 represented poor outcome of DLBCL. CDH23 is associated with essential biological functions and key molecules in DLBCL. CDH23 may play crucial roles in DLBCL tumorigenesis. Our results lay a foundation for further investigation of the role of CDH23 in DLBCL tumorigenesis.

Comprehensive Proteomic Analysis of Lysine Acetylation in Nicotiana benthamiana After Sensing CWMV Infection.

Protein lysine acetylation (Kac) is an important post-translational modification mechanism in eukaryotes that is involved in cellular regulation. To investigate the role of Kac in virus-infected plants, we characterized the lysine acetylome of Nicotiana benthamiana plants with or without a Chinese wheat mosaic virus (CWMV) infection. We identified 4,803 acetylated lysine sites on 1,964 proteins. A comparison of the acetylation levels of the CWMV-infected group with those of the uninfected group revealed that 747 sites were upregulated on 422 proteins, including chloroplast localization proteins and histone H3, and 150 sites were downregulated on 102 proteins. Nineteen conserved motifs were extracted and 51 percent of the acetylated proteins located on chloroplast. Nineteen Kac sites were located on histone proteins, including 10 Kac sites on histone 3. Bioinformatics analysis results indicated that lysine acetylation occurs on a large number of proteins involved in biological processes, especially photosynthesis. Furthermore, we found that the acetylation level of chloroplast proteins, histone 3 and some metabolic pathway-related proteins were significantly higher in CWMV-infected plants than in uninfected plants. In summary, our results reveal the regulatory roles of Kac in response to CWMV infection.

A virus-derived siRNA activates plant immunity by interfering with ROS scavenging.

Virus-derived small interference RNAs (vsiRNAs) not only suppress virus infection in plants via induction of RNA silencing but also enhance virus infection by regulating host defensive gene expression. However, the underlying mechanisms that control vsiRNA-mediated host immunity or susceptibility remain largely unknown. In this study, we generated several transgenic wheat lines using four artificial microRNA expression vectors carrying vsiRNAs from Wheat yellow mosaic virus (WYMV) RNA1. Laboratory and field tests showed that two transgenic wheat lines expressing amiRNA1 were highly resistant to WYMV infection. Further analyses showed that vsiRNA1 could modulate the expression of a wheat thioredoxin-like gene (TaAAED1), which encodes a negative regulator of reactive oxygen species (ROS) production in the chloroplast. The function of TaAAED1 in ROS scavenging could be suppressed by vsiRNA1 in a dose-dependent manner. Furthermore, transgenic expression of amiRNA1 in wheat resulted in broad-spectrum disease resistance to Chinese wheat mosaic virus, Barley stripe mosaic virus, and Puccinia striiformis f. sp. tritici infection, suggesting that vsiRNA1 is involved in wheat immunity via ROS signaling. Collectively, these findings reveal a previously unidentified mechanism underlying the arms race between viruses and plants.

Construction and biological characterization of an infectious full-length cDNA clone of a Chinese isolate of Wheat yellow mosaic virus.

Wheat yellow mosaic virus (family Potyviridae; genus Bymovirus), is an important soil-borne virus that causes serious economic losses in wheat. In this study, we constructed infectious cDNA clones of WYMV genomic RNAs under the control of 35S or SP6 promoter for versatile usage (agroinfiltration or in vitro RNA transcription). Our results showed that an Agrobacterium-mediated inoculation system enabled WYMV to infect the leaves of Nicotiana benthamiana without causing WYMV systemic infection. However, in vitro transcripts from infectious cDNA clones using the SP6 promoter promoted WYMV systemic infection of wheat plants, which was then developed for further assays. The optimal temperature for virus multiplication and systemic infection of wheat was 8 °C. Additionally, a synergistic effect between WYMV and Chinese wheat mosaic virus (CWMV) was also detected. This is the first report of the construction of a Chinese isolate of WYMV and should facilitate the investigation of viral pathogenesis.

Comparative proteomic analysis of Nicotiana benthamiana plants under Chinese wheat mosaic virus infection.

BACKGROUND: Chinese wheat mosaic virus (CWMV) is a severe threat to winter wheat and is transmitted by Polymyxa graminis. The mechanisms of interactions between CWMV and plants are poorly understood. In this study, a comparative proteomics analysis based on nanoliquid chromatography mass spectrometry (MS)/MS was conducted to characterize proteomic changes in plants responding to CWMV infection. RESULTS: In total, 2751 host proteins were identified, 1496 of which were quantified and 146 up-regulated and 244 down-regulated proteins were identified as differentially expressed proteins (DEPs). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that DEPs were most strongly associated with photosynthesis antenna proteins, MAPK signaling plant and glyoxylate and dicarboxylate metabolism pathways. Subcellular localization analysis predicted that more than half of the DEPs were localized in the chloroplast, an organelle indispensable for abscisic acid (ABA) synthesis. Our results suggest that CWMV infection interrupts normal chloroplast functions and decreases ABA concentrations in Nicotiana benthamiana. Further analysis showed that the ABA pathway was suppressed during CWMV infection and that ABA treatment induced plant hosts defenses against CWMV. CONCLUSIONS: We identified several candidate proteins expressed during CWMV infection, and the ABA pathway was strongly associated with responses to CWMV infection in N. benthamiana.

NbWRKY40 Positively Regulates the Response of Nicotiana benthamiana to Tomato Mosaic Virus via Salicylic Acid Signaling.

WRKY transcription factors play important roles in plants, including responses to stress; however, our understanding of the function of WRKY genes in plant responses to viral infection remains limited. In this study, we investigate the role of NbWRKY40 in Nicotiana benthamiana resistance to tomato mosaic virus (ToMV). NbWRKY40 is significantly downregulated by ToMV infection, and subcellular localization analysis indicates that NbWRKY40 is targeted to the nucleus. In addition, NbWRKY40 activates W-box-dependent transcription in plants and shows transcriptional activation in yeast cells. Overexpressing NbWRKY40 (OEWRKY40) inhibits ToMV infection, whereas NbWRKY40 silencing confers susceptibility. The level of salicylic acid (SA) is significantly higher in OEWRKY40 plants compared with that of wild-type plants. In addition, transcript levels of the SA-biosynthesis gene (ICS1) and SA-signaling genes (PR1b and PR2) are dramatically higher in OEWRKY40 plants than in the control but lower in NbWRKY40-silenced plants than in the control. Furthermore, electrophoretic mobility shift assays show that NbWRKY40 can bind the W-box element of ICS1. Callose staining reveals that the plasmodesmata is decreased in OEWRKY40 plants but increased in NbWRKY40-silenced plants. Exogenous application of SA also reduces viral accumulation in NbWRKY40-silenced plants infected with ToMV. RT-qPCR indicates that NbWRKY40 does not affect the replication of ToMV in protoplasts. Collectively, our findings suggest that NbWRKY40 likely regulates anti-ToMV resistance by regulating the expression of SA, resulting in the deposition of callose at the neck of plasmodesmata, which inhibits viral movement.

Wheat Yellow Mosaic Virus NIb Interacting with Host Light Induced Protein (LIP) Facilitates Its Infection through Perturbing the Abscisic Acid Pathway in Wheat.

Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly reliant on host factors to fulfill their infection. However, few host factors have been identified to participate in wheat yellow mosaic virus (WYMV) infection. Here, we demonstrate that wheat (Triticum aestivum) light-induced protein (TaLIP) interacts with the WYMV nuclear inclusion b protein (NIb). A bimolecular fluorescence complementation (BIFC) assay displayed that the subcellular distribution patterns of TaLIP were altered by NIb in Nicotiana benthamiana. Transcription of TaLIP was significantly decreased by WYMV infection and TaLIP-silencing wheat plants displayed more susceptibility to WYMV in comparison with the control plants, suggesting that knockdown of TaLIP impaired host resistance. Moreover, the transcription level of TaLIP was induced by exogenous abscisic acid (ABA) stimuli in wheat, while knockdown of TaLIP significantly repressed the expression of ABA-related genes such as wheat abscisic acid insensitive 5 (TaABI5), abscisic acid insensitive 8 (TaABI8), pyrabatin resistance 1-Llike (TaPYL1), and pyrabatin resistance 3-Llike (TaPYL3). Collectively, our results suggest that the interaction of NIb with TaLIP facilitated the virus infection possibly by disturbing the ABA signaling pathway in wheat.

The seven transmembrane domain protein MoRgs7 functions in surface perception and undergoes coronin MoCrn1-dependent endocytosis in complex with Gα subunit MoMagA to promote cAMP signaling and appressorium formation in Magnaporthe oryzae.

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of Gα subunits, thereby regulating G-protein mediated signal transduction. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, but not MoRgs8, couples with Gα MoMagA to undergo endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobiccues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.

[Clinical observation of 100 patients with malignant lymphoma treating with different preconditioning regimens followed by autologous hematopoietic stem cell transplantation].

This study was designed to compare the curative effect, prognosis and safety of different preconditioning regimens for patients who received autologous hematopoietic stem cell transplantation (AHSCT) for malignant lymphoma (ML). The clinical data of 100 ML patients (Sep 1992 to Aug 2010 in 307 Hospital) were retrospectively analyzed, and were divided into two groups by different preconditioning regimens: the high-dose chemotherapy preconditioning group and high-dose chemotherapy/radiotherapy preconditioning group. The overall survival (OS) rate, progress free survival (PFS) rate and adverse effect were analyzed. The results showed that until Feb 2011, the median follow-up was 33.5 months. All patients were engrafted and their hematopoiesis was reconstituted. The median time of WBC recovery up to > 1.0×1.0(9)/L in high-dose chemotherapy preconditioning group and high-dose chemotherapy/radiotherapy preconditioning group were (6.0 ± 0.4) d and (8.2 ± 0.4) d, platelet up to > 20.0×1.0(9)/L in two groups were (7.1 ± 0.8) d and (11.4 ± 2.5) d (P < 0.05). The 3-year OS rate of the two groups were 67.3% and 68.9%. 5-year OS rates of two groups were 62.8% and 60.6%, 10-year OS rates of two groups were 57.6% and 56.2% respectively; 3-year PFS of two group were 63.6% and 63.2%, 5-year of two group were 59.4% and 58.3%, 10-year of two group were 50.8% and 55.3% respectively (P > 0.05). Meanwhile, the incidence of fever, infection, bleeding, secondary cancer between two groups was not significant different (P > 0.05). It is concluded that the hematopoietic reconstitution of high-dose chemotherapy/radiotherapy preconditioning group is later than that of high-dose chemotherapy preconditioning group. However, there is no significant difference in curative effect and prognosis between the two groups.

[Retrospective analysis for 104 cases of early-stage Hodgkin's Lymphoma treated with different modality therapies].

This paper explored the curative effect of combined modality therapy and extended field radiotherapy for early-stage Hodgkin's Lymphoma. 104 cases of early-stage Hodgkin's Lymphoma from Jan 1987 to Dec 2010 in PLA Hospital 307 were retrospectively analyzed, including 76 cases in combined modality therapy group and 28 cases in extended field radiotherapy group, and the long-term efficacy and toxicity of two therapy modalities were evaluated. The results showed that the median survival time of 104 cases was 85.42 months, the complete remission rates of combined modality therapy and extended field radiotherapy groups were 72.4 and 71.4 respectively (P = 0.924); the overall response rates of combined modality therapy and extended field radiotherapy groups were 97.4 and 96.4 respectively (P = 0.779); the 5-year overall survival (OS) rates in the 2 groups were 89.5 and 89.1 respectively, and the 8-year OS rates of the 2 groups were 81.3 and 70.6. No statistical difference was found in above-mentioned 2 groups. Moreover, the 5-year progression free survival (PFS) rates of these 2 groups were 84.2 and 69.0 (P = 0.04), and 8-year PFS rates of these 2 groups were 80.0 and 55.5 (P = 0.04) respectively, the 5-year relapse rates of these 2 groups were 28.1 and 45.6 (P = 0.023) respectively. It is concluded that the combined modality therapy can raise the PFS rate and reduce the relapse rate as compared with extended field radiotherapy for early-stage Hodgkin's Lymphoma, but there is no difference in the overall survival rate between the 2 groups.

[Autologous peripheral blood stem cell transplantation combined with autologous bone marrow transplantation for treating refractory lymphoma].

This study was aimed to investigate the differences of therapeutic efficiencies, side effects and recovery rates of immune function in refractory lymphoma patients treated with autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT), autologous bone marrow transplantation (ABMT) and combination of APBHSCT with ABMT (APBHSCT + ABMT) by retrospective analysis, and to evaluate the merits and demerits of 3 kinds of transplantation for treatment of refractory lymphoma. 68 patients with malignant lymphoma were treated with autologous hematopoietic stem cells transplantation. Out of 68 patients 10 cases were treated with autologous bone marrow transplantation (ABMT), 46 cases were treated with autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT), and 12 cases were treated with autologous peripheral blood hematopoietic stem cells transplantation combined with autologous bone marrow transplantation (APBHSCT + ABMT). The results indicated that the therapeutic response rates and survival rates at 1, 3, 5 years for each transplant regimen were 90% and 75%, 57.1%, 33.3%; 86.4% and 74.4%, 54.2%, 38.1%; 83.3% and 72.7%, 55.6%, 40%. The times of ANC > or = 0.5 x 10(9)/L were 13, 11 and 9 days, times of platelet >/= 20 x 10(9)/L were 17, 14 and 10 days. The recovery rates of T cell subtypes in patients received ABMT, APBHSCT and APBHSCT + ABMT on 3 months, 6 months, 1 year were (0%, 33.3%, 60%), (10.8%, 32%, 73.9%), (27.3%, 55.6%, 85.7%) respectively. In conclusion, the efficacy and side effects of APBHSCT + ABMT as compared with ABMT and APBHSCT are roughly the same, but ABMT + APBHSCT can result in more rapid hematopoietic reconstitution and less restrictions with contributes to widen choice of transplant regimen for patients with alder age and impaired hematopoietic functions.

[Construction of a DNA vaccine against human B cell lymphoma].

AIM: To construct a DNA vaccine based on surface Ig V(H) gene of tumor cells in the human B-cell lymphoma biopsy tissue. METHODS: The V(H) gene fragment was amplified by RT-PCR using Ig superfamily primers. Also, the murine monocyte chemotactic protein 3(MCP-3) cDNA was cloned. The fusion gene fragment of MCP-3 gene with V(H) gene was constructed by recombinant PCR and then cloned into the eukaryotic expression vector pcDNA3.1 to construct the DNA vaccine plasmid pcDNA3.1/MCP-V(H). The vaccine plasmid was transiently expressed in the eukaryotic cell line COS-7. RESULTS: The DNA vaccine plasmid was successfully constructed and expressed in COS-7 cells in the form of fusion protein MCP-V(H). CONCLUSION: The DNA vaccine plasmid pcDNA3.1/MCP-V(H) is constructed and expressed successfully, which plays the foundation for further experimental research in animal model.

[The experimental study on idiotypic DNA vaccine against human B-cell lymphoma to induce antitumor immune response].

The purpose of this study was to evaluate whether the DNA vaccine containing idiotypic gene fragment of human B-cell lymphoma cell line Namalwa could elicit the specific anti-idiotypic immune response in vivo. The candidate gene fragment of the lymphoma cell, variable region of heavy chain (VH) of the membranous immunoglobulin, was amplified using Ig superfamily primers by means of RT-PCR. Also, the intact cDNA of murine monocyte chemoattractant protein (MCP-3) was cloned and used as the adjuvant molecular. The two gene fragments of VH and MCP-3 were fused together by 8aa linker peptide with recombinant PCR. Subsequently, the fusion gene fragment was cloned into eukaryonic expression vector pcDNA3.1 to construct DNA vaccine plasmid. Prior to the immunization, the transient transfection coupled with RT-PCR was performed to prove that the recombinant plasmid could express in eukaryonic cells in right way. Then two groups of mice were immunized by intramuscular injection with DNA vaccine and mock plasmid pcDNA3.1 respectively. Three times of injection were performed with 100 micro g plasmid respectively at the beginning of the experiment and 2, 4 weeks after the first injection for all the groups. FACS analysis was chosen to detect the antibodies recognizing lymphoma cells at different time following vaccination. The results demonstrated that specific anti-idiotypic antibody could be detected in the group of DNA vaccine immunized mice as early as eight weeks after the first immunization. Further test demonstrated that the anti-idiotypic antibody could maintain for at least twenty weeks with high titer. Anti-idiotypic antibodies were elicited in three of five mice of the DNA vaccine immunized group. The Abs of DNA vaccine immunized mice could only recognize Namalwa cell line instead of another unrelated human cell line A549. There is no cellular response detected in the DNA vaccine immunized mice. It is concluded that the DNA vaccine containing fused MCP3-VH sequence could elicit specific anti-idiotypic antibody against B-cell lymphoma in vivo and could be used in further study of DNA vaccine against B-cell lymphoma. The results would provide the basis for further studies and optimization of this therapeutic strategy on patients with B-lymphoproliferative disease.