Influence of the Structural Features of Carrageenans from Red Algae of the Far Eastern Seas on Their Antiviral Properties

The structural diversity and unique physicochemical properties of sulphated polysaccharides of red algae carrageenans (CRGs), to a great extent, determine the wide range of their antiviral properties. This work aimed to compare the antiviral activities of different structural types of CRGs: against herpes simplex virus type 1 (HSV-1) and enterovirus (ECHO-1). We found that CRGs significantly increased the resistance of Vero cells to virus infection (preventive effect), directly affected virus particles (virucidal effect), inhibited the attachment and penetration of virus to cells, and were more effective against HSV-1. CRG1 showed the highest virucidal effect on HSV-1 particles with a selective index (SI) of 100. CRG2 exhibited the highest antiviral activity by inhibiting HSV-1 and ECHO-1 plaque formation, with a SI of 110 and 59, respectively, when it was added before virus infection. CRG2 also significantly reduced the attachment of HSV-1 and ECHO-1 to cells compared to other CRGs. It was shown by molecular docking that tetrasaccharides—CRGs are able to bind with the HSV-1 surface glycoprotein, gD, to prevent virus–cell interactions. The revealed differences in the effect of CRGs on different stages of the lifecycle of the viruses are apparently related to the structural features of the investigated compounds.     

Anti HSV-1 Activity of Halistanol Sulfate and Halistanol Sulfate C Isolated from Brazilian Marine Sponge Petromica citrina (Demospongiae)

The n-butanol fraction (BF) obtained from the crude extract of the marine sponge Petromica citrina, the halistanol-enriched fraction (TSH fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate C (2), were evaluated for their inhibitory effects on the replication of the Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the viral plaque number reduction assay. The TSH fraction was the most effective against HSV-1 replication (SI = 15.33), whereas compounds 1 (SI = 2.46) and 2 (SI = 1.95) were less active. The most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. The anti-HSV-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into Vero cells, the virucidal effect on virus particles, and by the impairment in levels of ICP27 and gD proteins of HSV-1. In summary, these results suggest that the anti-HSV-1 activity of TSH fraction detected is possibly related to the synergic effects of compounds 1 and 2.     

Antiviral Potential of Sea Urchin Aminated Spinochromes against Herpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1) is one of the most prevalent pathogens worldwide requiring the search for new candidates for the creation of antiherpetic drugs. The ability of sea urchin spinochromes—echinochrome A (EchA) and its aminated analogues, echinamines A (EamA) and B (EamB)—to inhibit different stages of HSV-1 infection in Vero cells and to reduce the virus-induced production of reactive oxygen species (ROS) was studied. We found that spinochromes exhibited maximum antiviral activity when HSV-1 was pretreated with these compounds, which indicated the direct effect of spinochromes on HSV-1 particles. EamB and EamA both showed the highest virucidal activity by inhibiting the HSV-1 plaque formation, with a selectivity index (SI) of 80.6 and 50.3, respectively, and a reduction in HSV-1 attachment to cells (SI of 8.5 and 5.8, respectively). EamA and EamB considerably suppressed the early induction of ROS due to the virus infection. The ability of the tested compounds to directly bind to the surface glycoprotein, gD, of HSV-1 was established in silico. The dock score of EchA, EamA, and EamB was −4.75, −5.09, and −5.19 kcal/mol, respectively, which correlated with the SI of the virucidal action of these compounds and explained their ability to suppress the attachment and penetration of the virus into the cells.     

Intercellular trafficking of VP22, a herpes simplex virus type 1 tegument protein

BACKGROUND: The herpes simplex virus type 1 (HSV-1) tegument protein, VP22 has been reported to have the property of intercellular transport. The previous studies have shown that following expression of a fusion protein containing VP22; it spreads to every cell in a monolayer and concentrates in the nucleus. In spite of these reports, some studies have shown that VP22 trafficking and its nucleus accumulation is an artifact and no improvement in translocation of proteins fused to VP22 has been detected. METHODS: To better understand about VP22 translocation, VP22-GFP (Green Fluorescent Protein) vector was constructed and its nuclear accumulation, transportation to the nomtransfected cells and translocation between different cell types were studied by fluorescent microscope. RESULTS: VP22-fusion protein was detected in nontransfected cells which in some of them the fusion protein was shown in nucleus. CONCLUSION: The results demonstrated that VP22 can easily transport between different cells but nuclear accumulation of the protein is not common in all of the recipient cells.     

Immunogenicity Evaluation of a DNA Vaccine Expressing the Hepatitis C Virus Non-Structural Protein 2 Gene in C57BL/6 Mice

Backgrounds: Most of the hepatitis C virus (HCV) infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice. Methods: A plasmid encoding full-length HCV NS2 protein (non-structural protein 2) was generated and used to vaccinate mice. Negative control (an empty expression vector) was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte (CTL) activity and cytokine assay. Results: The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. Conclusion: DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses. Key Words: Hepatitis C virus (HCV), NS2 protein, DNA vaccine, IL-12     

Evaluation of bronchodilator and anti-anaphylactic activity of Myrica sapida

Background: Asthma is a chronic inflammatory disorder of the airways. The available treatment options have major limitations owing to low efficacy, associated adverse events and compliance issues. Therefore, the health burden of bronchial asthma is increasing globally at an alarming rate, providing a strong impetus for the development of new therapeutics. Myrica sapida is known traditionally in Ayurveda to possess anti-asthmatic activity. Hence, the present investigation was undertaken to evaluate the bronchodilator and anti-anaphylactic activity of the stem bark of Myrica sapida. Methods: Experimental models studied were acetylcholine induced bronchospasm in guinea pigs, egg albumin induced anaphylaxis in guinea pigs, in vitro studies on tracheal strip of egg albumin sensitized guinea pigs. Results: Treatment with ethanolic extract of M. sapida, 75 mg/kg, orally resulted in significant protection against acetylcholine aerosol induced bronchospasm and allergen induced anaphylaxis in guinea pigs. Ethanolic extract of M. sapida (75 mg/kg, p.o.) prevented the potentiation of responses and also produced a decrease in pD2 value of histamine and acetylcholine in guinea pig tracheal strip. Conclusion: These results suggest that M. sapida possesses bronchodilator activity, has potent inhibitory effect on immediate hyper-sensitivity reactions and decreases bronchial hyper-responsiveness.     

Antiviral activities and putative identification of compounds in microbial extracts from the Hawaiian coastal waters

Marine environments are a rich source of significant bioactive compounds. The Hawaiian archipelago, located in the middle of the Pacific Ocean, hosts diverse microorganisms, including many endemic species. Thirty-eight microbial extracts from Hawaiian coastal waters were evaluated for their antiviral activity against four mammalian viruses including herpes simplex virus type one (HSV-1), vesicular stomatitis virus (VSV), vaccinia virus and poliovirus type one (poliovirus-1) using in vitro cell culture assay. Nine of the 38 microbial crude extracts showed antiviral potencies and three of these nine microbial extracts exhibited significant activity against the enveloped viruses. A secosteroid, 5α(H),17α(H),(20R)-beta-acetoxyergost-8(14)-ene was putatively identified and confirmed to be the active compound in these marine microbial extracts. These results warrant future in-depth tests on the isolation of these active elements in order to explore and validate their antiviral potential as important therapeutic remedies.     

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM₁ binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. Key Words: Recombinant vaccine, Enterotoxigenic Escherichia coli (ETEC), cstH, eltB     

Cloning and expression of coxsakievirus B3 viral protein-1 in E. coli

BACKGROUND: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. METHODS: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. RESULTS: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. CONCLUSION: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.     

Sulfated Polysaccharides from Seaweed Strandings as Renewable Source for Potential Antivirals against Herpes simplex Virus 1

Herpes simplex virus 1 (HSV-1) remains a prominent health concern widespread all over the world. The increasing genital infections by HSV-1 that might facilitate acquisition and transmission of HIV-1, the cumulative evidence that HSV-1 promotes neurodegenerative disorders, and the emergence of drug resistance signify the need for new antiviral agents. In this study, the in vitro anti-herpetic activity of sulfated polysaccharides (SPs) extracted by enzyme or hot water from seaweeds collected in France and Mexico from stranding events, were evaluated. The anti-herpetic activity evaluation of the semi-refined-polysaccharides (sr-SPs) and different ion exchange purified fractions showed a wide range of antiviral activity. Among them, the sr-SPs from the Rhodophyta Halymenia floresii showed stronger activity EC₅₀ 0.68 μg/mL with SI 1470, without cytotoxicity. Further, the antiviral activity of the sr-SPs evaluated at different treatment schemes showed a high EC₅₀ of 0.38 μg/mL during the viral adsorption assays when the polysaccharide and the virus were added simultaneously, whilst the protection on Vero cell during the post-infection assay was effective up to 1 h. The chemical composition, FTIR and ¹H NMR spectroscopic, and molecular weights of the sr-SPs from H. floresii were determined and discussed based on the anti-herpetic activity. The potential utilization of seaweed stranding as a source of antiviral compounds is addressed.     

Antiviral lead compounds from marine sponges

Marine sponges are currently one of the richest sources of pharmacologically active compounds found in the marine environment. These bioactive molecules are often secondary metabolites, whose main function is to enable and/or modulate cellular communication and defense. They are usually produced by functional enzyme clusters in sponges and/or their associated symbiotic microorganisms. Natural product lead compounds from sponges have often been found to be promising pharmaceutical agents. Several of them have successfully been approved as antiviral agents for clinical use or have been advanced to the late stages of clinical trials. Most of these drugs are used for the treatment of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The most important antiviral lead of marine origin reported thus far is nucleoside Ara-A (vidarabine) isolated from sponge Tethya crypta. It inhibits viral DNA polymerase and DNA synthesis of herpes, vaccinica and varicella zoster viruses. However due to the discovery of new types of viruses and emergence of drug resistant strains, it is necessary to develop new antiviral lead compounds continuously. Several sponge derived antiviral lead compounds which are hoped to be developed as future drugs are discussed in this review. Supply problems are usually the major bottleneck to the development of these compounds as drugs during clinical trials. However advances in the field of metagenomics and high throughput microbial cultivation has raised the possibility that these techniques could lead to the cost-effective large scale production of such compounds. Perspectives on biotechnological methods with respect to marine drug development are also discussed.     

额种子蛋白体靶向表达口服重组胰岛素原载体构建及转化水稻

应用水稻种子生物反应器开发口服重组胰岛素原具有重要应用前景。通过分子设计保证重组胰岛素原在人体肠道内的自主加工成熟,根据水稻密码子偏爱性人工合成了霍乱毒素β亚基和人胰岛素原的融合基因(cholera toxin B subunit fused with human proinsulin,CTBIN),并在C末端添加内质网滞留信号KDEL。通过PCR技术从粳稻品种日本晴全基因组中克隆谷蛋白启动子及其信号肽序列pGluB1sig(GluB1 promoter and its signal peptide)用于驱动融合基因CTBIN的表达,插入载体pCAMBIA1302,构建了水稻种子蛋白体靶向表达口服重组胰岛素原的载体pCAMBIA1302-pGluB1sig-CTBIN-Nos。采用农杆菌介导法转化日本晴,获得了46株转基因水稻植株,Western杂交检测到CTB-人胰岛素原融合蛋白在水稻种子中表达。     

A Single Point Mutation within the Coding Sequence of Cholera Toxin B Subunit Will Increase Its Expression Yield

Background: Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. Methods: ctxB fragment from Vibrio cholerae O₁ ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F'' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB. Results: SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies. Conclusion: Point mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield. Key Words: Escherichia coli, Point mutation, Cholera toxin B subunit (CTB), Protein expression     

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Background: Herpes simplex virus type-1 (HSV-1) establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3''-diaminobenzidine (DAB) substrate. Methods: Eight-week-old male BALB/c mice were inoculated via the eye by 10⁴ plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. Results and Conclusion: The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia. Key Words: Herpes simplex virus-1, Latency, In situ PCR, two-round PCR, Trigeminal ganglia     

Design of a Novel Recombinant Multi-Epitope Vaccine against Triple-Negative Breast Cancer

Background:TNBC is determined by the absence of ERBB2, estrogen and progesterone receptors’ expression. Cancer vaccines, as the novel immunotherapy strategies, have emerged as promising tools for treating the advanced stage of TNBC. The aim of this study was to evaluate CEA, MTDH, and MUC-1 proteins as vaccine candidates against TNBC. Methods:In this research, a novel vaccine was designed against TNBC by using different immunoinformatics and bioinformatics approaches. Effective immunodominant epitopes were chosen from three antigenic proteins, namely CEA, MTDH, and MUC-1. Recombinant TLR4 agonists were utilized as an adjuvant to stimulate immune responses. Following the selection of antigens and adjuvants, appropriate linkers were chosen to generate the final recombinant protein. To achieve an excellent 3D model, the best predicted 3D model was required to be refined and validated. To demonstrate whether the vaccine/TLR4 complex is stable or not, we performed docking analysis and dynamic molecular simulation. Result:Immunoinformatics and bioinformatics evaluations of the designed construct demonstrated that this vaccine candidate could effectively be used as a therapeutic armament against TNBC. Conclusion:Bioinformatics studies revealed that the designed vaccine has an acceptable quality. Investigating the effectiveness of this vaccine can be confirmed by supplementary in vitro and in vivo studies.Key Words: Adjuvants, Triple-negative breast cancer, Vaccine     

Chitosan and Kappa-Carrageenan Vaginal Acyclovir Formulations for Prevention of Genital Herpes. In Vitro and Ex Vivo Evaluation

Vaginal formulations for the prevention of sexually transmitted infections are currently gaining importance in drug development. Polysaccharides, such as chitosan and carrageenan, which have good binding capacity with mucosal tissues, are now included in vaginal delivery systems. Marine polymer-based vaginal mucoadhesive solid formulations have been developed for the controlled release of acyclovir, which may prevent the sexual transmission of the herpes simplex virus. Drug release studies were carried out in two media: simulated vaginal fluid and simulated vaginal fluid/simulated seminal fluid mixture. The bioadhesive capacity and permanence time of the bioadhesion, the prepared compacts, and compacted granules were determined ex vivo using bovine vaginal mucosa as substrate. Swelling processes were quantified to confirm the release data. Biocompatibility was evaluated through in vitro cellular toxicity assays, and the results showed that acyclovir and the rest of the materials had no cytotoxicity at the maximum concentration tested. The mixture of hydroxyl-propyl-methyl-cellulose with chitosan- or kappa-carrageenan-originated mucoadhesive systems that presented a complete and sustained release of acyclovir for a period of 8–9 days in both media. Swelling data revealed the formation of optimal mixed chitosan/hydroxyl-propyl-methyl-cellulose gels which could be appropriated for the prevention of sexual transmission of HSV.     

Nutritional and Chemical Composition and Antiviral Activity of Cultivated Seaweed Sargassum naozhouense Tseng et Lu

Sargassum naozhouense is a brown seaweed used in folk medicine and applied for thousands of years in Zhanjiang, Guangdong province, China. This study is the first time to investigate its chemical composition and antiviral activity. On the dry weight basis, this seaweed was constituted of ca. 35.18% ash, 11.20% protein, 1.06% lipid and 47.73% total carbohydrate, and the main carbohydrate was water-soluble polysaccharide. The protein analysis indicated the presence of essential amino acids, which accounted for 36.35% of the protein. The most abundant fatty acids were C14:0, C16:0, C18:1 and C20:4. The ash fraction analysis indicated that essential minerals and trace elements, such as Fe, Zn and Cu, were present in the seaweed. IR analysis revealed that polysaccharides from cultivated S. naozhouense may be alginates and fucoidan. The polysaccharides possessed strong antiviral activity against HSV-1 in vitro with EC₅₀ of 8.92 μg/mL. These results demonstrated cultivated S. naozhouense has a potential for its use in functional foods and antiviral new drugs.     

Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2)

Background: RNA-binding motif gene on Y chromosome (RBMY), a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. Methods: Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. Results: Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. Conclusion: The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Key Words: Protein isoforms, Spermatogenesis, Male infertility     

建兰花叶病毒CP基因的原核表达及抗血清制备

通过间接酶联免疫检测和电镜观察对从福建省漳州市采集的卡特兰病样进行检测,证明样品感染了建兰花叶病毒。设计一对特异性引物,扩增并克隆病毒分离物的外壳蛋白基因,随后将目的基因插入pET-29a（＋）中构建相应的原核表达载体。目的蛋白经诱导表达及纯化后免疫家兔并获得了特异性抗血清。Westernblot检测结果表明,抗血清与诱导表达的CyMV外壳蛋白发生特异性反应。间接酶联免疫法检测结果表明,抗血清可检测病汁液的最低稀释度达1∶51200,最佳工作浓度为1∶1000,病汁液灵敏度为0.39mg/mL,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应。     

日粮能量和蛋白水平对瘦肉型生长育肥猪生长性能的影响

选取60±1.5 kg、90.0±1.5 kg杜长大(D×L×Y)生长育肥猪48头(公母比例为1∶1),通过对平均采食量(ADFI)、平均日增重(ADG)和料重比(F/G)进行比较分析,考察蛋白和能量水平对其生长性能的影响。结果表明:日粮能量和蛋白水平对60～90 kg瘦肉型生长育肥猪ADG、ADFI和F/G均有显著影响(P<0.05);日粮能量和蛋白水平对90～120 kg生长育肥猪ADG的影响不显著(P>0.05),对ADFI、F/G影响显著(P<0.05)。60～90 kg、90～120kg阶段杜长大生长育肥猪适宜的能量和蛋白水平分别为13.90 MJ.kg-1、15.90%和14.62 MJ.kg-1、14.90%;多元回归分析得到90～120 kg瘦肉型生长育肥猪DEI、CPI与ADG的回归方程为:ΔW=-12.772DEI+23.467CPI+351.156(R2=0.881,P<0.05)。