Abstract:Abstract： Bacteria have long been considered as the key driver of ammonia oxidation on earth. This concept has been challenged recently by the discovery of chemolithoautotrophic isolate of ammonia-oxidizing archaeon in marine. The relative contribution of bacteria and archaea to ammonia oxidation is essential for our understanding of global nitrogen cycle. Recent study suggested a key role of archaeal ammonia oxidation in the marine nitrogen cycle. Our work however revealed the predominace of bacterial ammonia oxidation in agricultural soil. From the biogeochemical perspective, here we summarized the discovery, progress and prospect of archaeal ammonia oxidation. Of great interest in the future would be to elucidate the metabolisms of ammonia-oxidizing archaeon in natural environment and the underlying mechanism that leads to the physiological divergence of ammonia oxidizers.

Abstract:Abstract: Microbial eco-systems metabolic networks (MEMNs) are ubiquitous in nature and are the major drivers of biosphere processes. Here we try to define MEMNs and describe their significances in microbial ecology studies. We also review recent progresses and approaches of MEMNs including highlights of the major discoveries in these studies based on mixed-cultures, high-throughput sequencing and microarray data, computational approaches, and metabolic reconstruction. In addition, we discuss the potential shortcomings of current approaches and propose that the time is ripe to study the energy-based microbial metabolic networks in bioremediation systems, especially under the anaerobic conditions, which will reclaim the wastes to resources.

Abstract:Abstract：Oxygenases are common tailoring enzymes involved in the biosynthesis of aromatic polyketides, they are critical determinants of the final structure of aromatic polyketides. Here we review the structures and functions of several classes of oxygenases found in the biosynthetic gene clusters of aromatic polyketides. We used the oxygenases (JadFGH) in jadomycin biosynthesis as examples to elucidate the complex roles these oxygenases play and discussed the future prospects of using oxygenases in combinatorial biosynthesis.

Abstract:Abstract: Strain 36R-2-1B was isolated from Allium fistulosum and identified as Streptomyces spp., harboring a ~280 kb linear plasmid designated pYY8L. [Objective] Cloning, sequencing and analysis of telomere and internal replication origin of pYY8L. [Methods] pYY8L telomere was cloned by a modified procedure ─“alkaline treatment and enzyme digestion in gel”. The internal replication origin of pYY8L was cloned by construction of a cosmid library and then sub-cloning. [Results] A ~280 kb DNA band (pYY8L) of strain 36R-2-1B was detected by pulsed-field gel electrophoresis. The 152-bp telomere, containing six small palindromes and potentially forming complicated secondary structure, was cloned and sequenced. The replication origin of pYY8L was initially cloned on a cosmid and then sub-cloned as a 4891-bp autonomous replication sequence. This sequence was predicted to contain six genes, two of them resembling replication genes of Streptomyces linear plasmid SCP1, but their adjacent iterons were different. [Conclusion] New telomere and internal replication origin of Streptomyces linear plasmid pYY8L were cloned and identified. This is the first example of report on telomere and replication genes of linear plasmid from endophytic Streptomyces.

Abstract:Abstract: [Objective] Comparative genomic study revealed mutations at the promoter regions of pabB and lpdA in avirulent Mycobacterium tuberculosis strain H37Ra compared to its virulent counterpart strain H37Rv. LacZ reporter system was used to test whether those mutations will affect promoter activity and its potential relationship to virulence attenuation of H37Ra. [Methods] Promoter regions of pabB and lpdA were predicted by the “Neural Network Promoter Prediction” method (http://www.fruitfly.org/seq_tools/promoter.html). Promoter sequences were PCR amplified and cloned into mycobacterial promoterless probe vector pMC210. Resultant recombinant plasmids were transformed into M. smegmatis by electroporation. The transcription activity of lacZ under the control of cloned promoters were monitored by Quantitative Real-Time RT-PCR. [Results] Quantitative Real Time PCR results showed that the promoter activity of H37Ra pabB was six times more than that of H37Rv pabB (p < 0.05), while the promoter activity of the H37Rv lpdA was two fold of that of H37Ra lpdA (p < 0.05). [Conclusion] The mutations in pabB and lpdA promoters affect its expression activity. The T-A mutation in lpdA promoter may be related to virulence attenuation of H37Ra.

Abstract:Abstract: [Objective] p48 (ac103) gene is highly conserved in baculovirus, implying that p48 might play a fundamental role in the life cycle of baculovirus.[Methods] In this study, We studied the expression of p48 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)---the type baculovirus species. With Bac-to-Bac system, we constructed two p48 repair viruses, in which HA-tag was fused to C-terminus and N-terminus of P48, respectively. To examine the effect on occlusion body morphogenesis and to facilitate examination of virus infection, the green fluorescence protein (gfp) gene and polyhedrin (polh) gene were also inserted into the recombinant viruses. Sf9 cells were transfected with each bacmid constructed, and the supernatants containing the budded viruses (BVs) were used to infect Sf9 cells. At the indicated time points, cells were harvested and used for SDS-PAGE and Western blot analysis. The expression of fusion protein was detected with the monoclonal antibody to the HA-epitope. [Results] Western blot analysis indicated that a specific 43 kDa protein was detected at 12 hours postinfection (hpi) and remained detectable up to 96 hpi in cells infected with p48 repair virus with hemagglutinin (HA)-tag fused in C-terminus. Meanwhile, another protein of 26 kDa was also detected from 12 hpi to 96 hpi. However, no signals were detected in cells infected with p48 repair virus with HA-tag fused in N-terminus till 96 hpi. [Conclusion] The result indicated that p48 is a late gene and expressed in late infection. P48 might be cleaved in N-terminal when it is expressed in insect cells.

Abstract:Abstract: [Objective] A high quality library of marine microbes and their associated natural products is critical for successful drug discovery. In this research a method to assess the quality of our marine microbial natural product library through screening for novel enediyne-like compounds from this library was set up. [Methods] A high throughput screening assay based on the unique DNA-damage activity of enediyne-like compounds has been constructed and our marine microbial natural product library was screened. Because the polyketide synthase responsible for the biosynthesis of enediyne core is a conserved iterative type I polyketide synthase, a sequence-based screening method was built to get the strains that harbored enediyne biosynthesis gene clusters. [Results] Through activity-based screening, a positive hit from our marine natural product library was acquired. 16S rRNA analysis has revealed that this strain-LS481 was 99% similar to Micromonospora chersina DSM 44151T that could produce enediyne compound-Dynemicins. The crude extract of LS481 showed similar characteristics with Dynemicin A. In addition, two strains, MS098 and LS2004, were acquired through sequence-based screening. The 16S rRNA of MS098 was 100% the same as Streptomyces griseus NBRC 13350, and LS2004 was most close to Streptomyces vinaceus NBRC 13425T and Streptomyces cirratus NRRL B-3250T. [Conclusion] We can successfully isolate enediyne compounds through the activity-based screening and assess the potential of producing enediyne compounds through sequence-based screening.

Abstract:Abstract: [Objective] In this study, we investigated the cross-protection of Lactobacillus casei ATCC? 393TM under multi-stress conditions. [Methods] Cells pre-adapted to mild conditions (heat, H2O2, acid or bile salts) were then treated at lethal temperature (>60 oC) or hydrogen peroxide stress (>5 mmol/L). Furthermore, the changes of survival rate, intracellular pH and membrane fatty acid under lethal conditions with or without acid adaption were compared. [Results] The cross-protection in Lactobacillus casei ATCC 393TM were affected by different stress conditions cell encountered. Acid pre-adaption, especially hydrochloride treatment, would increase the resistance of cells to lethal heat and peroxide stresses significantly, with the survival rate of 305-fold and 173-fold, respectively. Further study suggested that the effect of acid pre-adaption might be related to the regulation on intracellular pH and the saturation of cell membrane. [Conclusion] In this study, hydrochloride adaption was the best inducer for the cross-protection of Lactobacillus casei ATCC 393TM to maintain relatively stable physiological status of cells. The results above supplied a novel way to investigate the relationship between different protective mechanisms in L. casei under different kinds of stresses.

Abstract:Abstract: [Objective] Hydroxylamine is an important intermediate product of anammox. This study was focused on the characteristics of hydroxylamine and nitrite conversions by anammox enrichment. [Methods] The changes of nitrogenous substrates and related products with time were measured using batch tests with anammox enrichment as inoculum. [Results] Since hydroxylamine didn’t react with nitrite in uninoculated control culture, these two compounds were chemically stable. Both of them decreased with time in anammox enrichment inoculated cultures, in which ammonia as intermediate product would be produced and converted with the maximum concentration being 0.338 mg/L. The total nitrogen concentration decreased from 4.694 mmol/L to 0.812 mmol/L with conversion rate 82.7 % in the end. When hydroxylamine and nitrite concentrations were about 2.5 mmol/L respectively, the maximum specific sludge conversion rates of hydroxylamine was 0.535 mmol/(gVSS.h), which was 1.81 times bigger than that of ammonia in ammonia reaction system; the maximum specific sludge rate of total nitrogen was slightly higher than that in ammonia reaction system. When hydroxylamine concentration increased to 5.0 mmol/L, the hydroxylamine and nitrite conversion rates promoted by 26.7% and 120.7% respectively; and the maximum ammonia accumulated was 1.810mmol/L. When nitrite concentration increased to 5.0 mmol/L, the hydroxylamine and nitrite conversion rates promoted by 6.9% and 9.0% respectively; and the maximum ammonia accumulated was 0.795 mmol/L. [Conclusion] Anammox enrichment was capable of converting hydroxylamine and nitrite simultaneously and had the higher conversion rate of hydroxylamine than ammonia conversion rate. Hydroxylamine and nitrite conversion rates were less affected by increase in nitrite concentration, but more significantly influenced by increase in hydroxylamine. The maximum ammonia concentration accumulated would rise as the result of increasing both hydroxylamine and nitrite. The result of experiment was consistent with pathway model presented by van de Graaf AA.

Abstract:Abstract: [Objective] In order to deeply understand the role of the entomopathogenic fungal chitinase in invasion and pathogenic process, we cloned and expressed a chitinase gene from Nomuraea rileyi CQ strain, and detected chitinase activity in recombinant Pichia pastori fermentation supernatant. [Methods] Specific primers were used to amplify the complete sequence of chitinase gene and open reading frame (ORF) from genomic DNA of N. rileyi Cq Strain extracted by CTAB. The fragment of ORF was linked with vector plasmid pPIC9K to construct the pPIC9K-chit1 expressing vector and transferred into Pichia pastori. The recombinant yeast P. pastori was screened by 1.5mg/L G418 and PCR check. Activity of chitinase in recombinant P. pastori fermentation liquid was verified by transparent zone test and OD540 determination, and the molecular weight of chitinase was analyzed by SDS-PAGE. [Results] The complete sequence length of chitinase gene of N. rileyi CQ strain is 2756bp (GenBank accession number EU795711) that contains a 1827bp of ORF chit1, a 76bp uncoding region at the 5’end, a 240bp uncoding region at the 3’end, and 3 introns. The ORF chit1 encoding 424 amino acids of chitinase precursor, and the theoretical restriction site of single peptide is between Gly(20) and Leu(21). Activity of chitinase expressed by recombinant P.pastoris increased with fermentation time, and reached the peak of 482.5U/100μL at 72h. Hydrolysis test of chitin showed clear transparent zone at the 1% colloidal chitin plate. SDS-PAGE analysis suggested that the molecular weight of chitinase was 41.0 kDa. [Conclusion] This research cloned the chitinase gene Chit1 from N. rileyi CQ strain and expressed in recombinant P. pastori successfully. It has great significance for in-depth study on the infection and lethal mechanism of pathogenic fungi to insects.

Abstract:Abstract: [Objective] In order to reveal the relationships of compositions and content of pigment in pigment-protein complexes (PPC) and hydrogen photoevolution from anoxygenic phototrophic bacteria. [Methods] We isolated and identified pigment-protein complexes using a separation strategy of subsequent fractionated ammonium-sulfate precipitation, ion exchange column chromatography, absorption spectra and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) from hydrogen-producing Rhodobacter azotoformans R7. We investigated the characterizations of the peripheral light-harvesting complex (LH2) with an unusual absorption spectrum by surface enhanced laser desorption / ionization time of flight mass spectrometry (SELDI-TOF-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and fluorescence spectra. [Results] We acquired three types of PPC, the reaction center and core light-harvesting complex (RC-LH1) and two kinds of LH2, from strain R7 incubated anaerobically in the light. The two LH2 showed the different absorption spectra, one of them displayed unusual absorption spectrum with the maximum absorption band at 423 nm. The unusual LH2 consisted of two kinds of protein subunits with the molecular weight of 5556.8 Da and 5697.8 Da and carotenoid of spheroidene series with the molecular weight of 562 Da. It was also capable of transferring energy from carotenoid to bacterialcholorophyll and from B800 bacterialcholorophyll to B850 bacterialcholorophyll. [Conclusion] Rhodobacter azotoformans R7 with hydrogen-producing capacity could photosynthesize two types of LH2 under anaerobically in the light, one of them presented novel spectral property.

Abstract:Abstract: [Objective]Interleukin-18(IL-18) is a proinflammatory cytokines that plays a key role of immune regulation through activating the Th 1 cell and NK cell secreting interferon-γ(IFN-γ). IL-18-binding proteins(IL-18BP) secreted in human and mouse can antagonise the activity of IL-18. The homologue gene of IL-18BP from fowlpox virus genome is predicted and identified of its expressed protein，which is helpful to use as an antagonist to IL-18 guiding disease. [Methods] The cIL-18BP gene was isolated from the genome of fowlpox vaccine virus by PCR through a pair of special primers and cloned into vectors pPICZαA，then expressed in yeast GS115. The biologic activity of recombinant proteins binding with cIL-18 was measured, inhibiting the activity of cIL-18 enhancing relative cells secreting IFN-γ was confirmed through ELISA. [Results] The cIL-18BP gene was cloned from fowlpox virus and acquired high performance expression in yeast systems induced with methanol identified by SDS-PAGE. Recombinant cIL-18BP purified can bind specifically with recombinant cIL-18 determined by ELISA test. cIL-18BP can antagonise the activity of cIL-18 with determining the IFN-γ concentration secreting in PBMCs and MSB1 cells stimulated by cIL-18 respectively. [Conclusion] The experiment indicates that recombinant cIL-18BP can inhibit the activity of cIL-18 stimulating related immune cells secreting IFN-γ. Deletion of the cIL-18BP from virus may improve the safety and immunogenicity of fowlpox virus vaccine.

Abstract:Abstract：[Objective] To determine if the fowlpox virus (FPV) ORF73 or ORF214 gene encoding protein has the function of IL-18 binding protein, and assess the role of that ORF73 or ORF214 gene played in the regulation of immune response. [Methods] Recombinant fowlpox virus (rFPV) vector-based rFPVLP-△73LRH5A or rFPVLP-△214LRH5A, expressing avian influenza haemagglutinin gene (H5A) with ORF73 or ORF214 gene deletion, was constructed respectively. The parental recombinant virus expressing avian influenza haemagglutinin （rFPVLP- 12LSH5A）was used as the control virus. In this study, the production of IFN in vitro by splenocytes and peripheral blood mononuclear leukocytes (PBML) of SPF chickens stimulated with rFPVs was detected, and the immune efficacy，antibody responses，ratio of CD4+/CD8+ T lymphocyte and multiplication capacity of PBML induced by the rFPVs vector-based rFPVLP-△73LRH5A, rFPVLP-△214LRH5A and rFPVLP- 12LSH5A vaccine were evaluated in SPF chickens. [Results] The level of IFN production from splenocytes stimulated with rFPVLP-△73LRH5A and rFPVLP-△214LRH5A was significantly higher than that with rFPVLP-12LSH5A in vitro, whereas the ratio of CD4+/CD8+ T lymphocyte in rFPVLP-△73LRH5A and rFPVLP-△214LRH5A groups was significantly lower than in rFPVLP- 12LSH5A groups after 10 days post immunization (dpi). These rFPVs stimulated the proliferation of PBML without significant difference. All chickens immunized with rFPVLP-△73LRH5A, rFPVLP-△214LRH5A or rFPVLP- 12LSH5A produced HI antibodies against H5 AIV HA antigen, and rFPVLP-△214LRH5A induced significantly lower HI titer than rFPVLP- 12LSH5A after 14 dpi. All immunized chickens were fully protected against lethal challenge of H5N1 AIV. [Conclusion] The results show that ORF73 and ORF214 encoding proteins blocks induction of IFN, suggesting that they are provided with IL-18BP functionality. Despite of decreasing humoral response and cell-mediated immune response, rFPV with deleted FPV73 or FPV214 gene could induce the effective efficacy in SPF chickens.

Abstract:Abstract: [Objective] To construct prokaryotic fusion gene expression vector pET-30a/ltB-porB, express the recombinant fusion protein LTB-PorB and analyze the immunocompetence of the recombinant fusion protein in female BALB/c mice through intranasally immunization. [Methods] B subunit of Escherichia coli heat-labile enterotoxin (LTB) and Neisseria gonorrhoeae Porin B (PorB)fusion gene, LTB gene and PorB gene were cloned into prokaryotic vector pET-30a. The recombinants were identified by Polymerase Chain Reaction(PCR), enzyme digestion and DNA sequencing, and then expressed efficiently in Escherichia coli BL21 in the form of inclusion bodies. The renatured recombinant proteins had antigenicity, which was confirmed by Western blot. Female BALB/c mice were inoculated with renatured recombinant proteins without endotoxin through intranasally immunization at the days 0, 14, 28. Next, humoral immunoresponse and cellullar immunologic response were detected in female BALB/c mice by enzyme linked immunosorbent assay(ELISA) and methyl thiazolyl tetrazolium(MTT) colorimetric assay. [Results] The level of PorB specific sIgA in genital tract and IgG in serum shown upward trend along with the days post innoculation in LTB-PorB group, A450 of sIgA in LTB-PorB group was 0.66 at the day 42, which was significantly higher than controls (P<0.01), and the titer was up to 1:1280. A450 of serum IgG in LTB-PorB group was 0.60 at the day 28, which was significantly higher than the LTB and the Solution Buffer controls (P<0.01), and the titer was up to 1:2560. However, the IgG between LTB-PorB group and PorB control(A450:0.57)had no significant difference (P>0.05). Stimulation index of the splenic lymphocyte in LTB-PorB group was significantly higher than the LTB and the Solution Buffer controls (P<0.05). But the level of IFN-γ induced by splenic lymphocyte between LTB-PorB group and controls had no significant difference (P>0.05). [Conclusion] The recombinant fusion protein LTB-PorB could induce high level of humoral immunoresponse and slightly cellullar immunologic response in female BALB/c mice through intranasally immunization. For the first time to our knowledge, the mucosal adjuvant LTB could assist PorB to induce high level of mucosal immune response in the genital tract mucosa of mice.

Abstract:Abstract: [Objective] Establishment of DNA fingerprinting in Phytophthora sojae and an analysis of genetic relationship of Heilongjiang and Xinjiang populations. [Methods] Bioinformatics tools were used to search repetitive sequences in P. sojae and Southern blot analysis was employed for DNA fingerprinting analysis of P. sojae populations from Heilongjiang and Xinjiang using the identified repetitive sequence. [Results] A moderately repetitive sequence was identified and designated as PS1227. Southern blot analysis indicated 34 distinct bands ranging in size from 1.5 kb-23 kb, of which 21 were polymorphic among 49 isolates examined. Analysis of single-zoospore progenies showed that the PS1227 fingerprint pattern was mitotically stable. DNA fingerprinting showed that the P. sojae isolates HP4002, SY6 and GJ0105 of Heilongjiang are genetically identical to DW303, 71228 and 71222 of Xinjiang, respectively. [Conclusion] A moderately repetitive sequence designated PS1227 which will be useful for epidemiology and population biology studies of P. sojae was obtained, and a PS1227-based DNA fingerprinting analysis provided molecular evidence that P. sojae in Xinjiang was likely introduced from Heilongjiang.

Abstract:Abstract：[Objective] To study the endophyte species of Achnatherum inebrians and to screen strains which have insecticidal activity to cotton insect.[Methods] We isolated endophytic from roots, stems, leaves and seeds of health A. inebrians by grinding separation method and identified by a dual approach of morphological and physiological observation and 16S rDNA gene (for bacteria) and ITS sequence (for fungi) based molecular identification. Then, those endophytes were inoculated into liquid media for fermentation and the crude extracts were used to insecticidal activities tests by slide disc immersion and nebulization methods.[Results] A total of 89 bacteria species that classified into 8 genera of Bacillus, Streptomyces, Corynebacterium, Phyllobacterium, sphingomonnas, Paenibacillus, Pseudomonas, Acinetobacter and 2 fungi of Claviceps purpure and Claviceps Chaetomium, were isolated. Of them, the strain Streptomyces rochei (GA) and Claviceps purpurea (PF-2) were showed more than 85% of mortality to cotton aphis, after primary and second screenings. [Conclusion] Two strains of PF-2 and GA associated within the A. inebrians had significant insecticidal activity to cotton aphis(Aphis gossypii), which may provide a new biological resource to explore new microbial insecticide.

Abstract:Abstract: [Objective] New wild strains, which were endowed with an effective depilation as their abilities to produce keratinase, were isolated to develop the bio-deliplatory agent in the sulfur-free tanning. [Methods] A polyphase screening method was used to obtain the high-yielding wild strains. In this method, the sewage sample was obtained from the specific circumstance where the raw hides were stored, and then enriched by adding the defatted wool power as an inducer in the medium. Furthermore, the strains were screened via assessing the ability of the depilation of their fermentation broth. Finally, the isolated strain was identified according to their morphological features, physiological and biochemical characteristics, automatic identification by Biolog system as well as phylogenetic analysis of 16SrDNA sequences. [Results] A higher activity and remarkable dehairing capability with sulfur-free was obtained. The results of identification indicated that the strain X-47 was the most closely related to Bacillus licheniformis, so it was named Bacillus licheniformis X-47. [Conclusion] The wild strain Bacillus licheniformis X-47 was isolated successfully by the method of polyphase screening. It had a high keratinase-producing activity, high efficient of depilation and weak effect on collagen, which suggested that it has a considerable potential of developing the sulfur-free bio-depilation product.

Abstract:Abstract: [Objective] Bacterial strain named M-1 was isolated from the diseased Sciaenops ocellatus with ulceration, to further analyze the phylogenetics of the bacterial strain. [Methods] We adopted the characteristics of morphology and physiology, accompanied with the sequence comparison of 16S rRNA and HSP60 genes. [Results] Strain M-1 was identified as Vibrio splendidus. 16SrRNA gene of strain M-1 and Vibrio splendidus(AJ874361) had nearest kinship, and the analysis of HSP60 gene sequence indicated that strain M-1 togethered with Vibrio (EU653883、AY837440)in phylogenetic tree. [Conclusion] The results suggested that strain M-1 was a member of Vibrio splendidus.

Abstract:Abstract：[Objective] Our aim in this study was to develop a whole-genome DNA microarray of Vibrio parahaemolyticus, build up a method of microarray hybridization and carry on microarray quality evaluation. [Methods] Based on the genomic sequences of V. parahaemolyticus, we chose a total number of 4770 genes, amplified them by PCR with specific primers, purified the PCR products and printed them onto glass slides. We performed two sets of hybridizations by the method of two-fluorescence comparative hybridization to evaluate the microarray quality, followed by PCR method to validate parts of microarray results. [Results] Microarray hybridization results were completely consistent with theory expectations and PCR verification results. [Conclusion] We successfully developed a batch of good quality whole-genome DNA microarrays of V. parahaemolyticus, built up a method of microarray-based comparative genomic hybridization of V. parahaemolyticus and a set of microarray data analysis standard method.

Abstract:Abstract：[Objectives] To evaluate the humoral and cellular immune responses in C57BL/6 mice induced by pcDNA3.1(+)/Lpp20-IL2 for further development of DNA vaccine against H.pylori infection. [Methods] The eukaryotic expression vector pcDNA3.1(+)/Lpp20-IL2 was constructed and then transfected into HeLa cells using liposome. Western blot was used to verify the expression of Lpp20 antigen gene in HeLa cells. Then five groups of 6-week-old C57BL/6 mice were immunized intramuscularly with either of the following vaccines: pcDNA3.1(+)/Lpp20-IL2, pcDNA3.1(+)/Lpp20, pcDNA3.1(+)/IL2, mock plasmid pcDNA3.1(+), or PBS four times at 1-week interval. Six weeks after the last injection, the specific IgG in the sera of C57BL/6 mice and the cytokine content of IFN-γ and IL4 in mice spleen lymphocyte culture medium after stimulation by recombinant Lpp20（rLpp20）was tested quantitatively by ELISA,respectively. The proliferation of spleen cells was measured by MTT assay. Lpp20-IL2 fusion protein in mouse muscular tissue was detected by immunofluorescence histochemistry. [Results] The eukaryotic expression recombinant pcDNA3.1(+)/Lpp20-IL2 was successfully constructed and could be expressed in HeLa cells . The significant specific antibody titers were detected by ELISA in pcDNA3.1(+)/Lpp20-IL2 DNA vaccine groups and the highest titer was 1:4096 after eight weeks. The cytokines content of IFN-γ and IL4 in cultural supernatant of spleen lymphocytes from mice immunized with pcDNA3.1(+)/Lpp20-IL2 increased significantly.After stimulated by corresponding antigen, the stimulation index of pcDNA3.1(+)/Lpp20-IL2 and pcDNA3.1(+)/Lpp20 group were higher than that of control groups(P<0.01). Lpp20-IL2 fusion protein could be expressed in mouse muscular tissue. [Conclusions] We have successfully constructed the recombinant eukaryotic expression plasmid pcDNA3.1(+)/Lpp20-IL2 and the Lpp20-IL2 fusion protein could be effectively expressed in HeLa cells.Both pcDNA3.1(+)/Lpp20-IL2 and pcDNA3.1(+)/Lpp20 DNA vaccines could induce strong cellular immunity and humoral immunity in C57BL/6 mice,and the former could induce more powerful cellular immunologic response.