Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

IDENTIFICATION OF PATHOGENIC LEPTOSPIRES BY RECOMBINANT DNA PROBES

Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector PUC9. Recombinant plasmids which have hornologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid,designated PCX7, could detect 1.7 kb fragment of strain 017, 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and hoptonema illini. The recombinant plasmid PCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.

DNA存储技术:挑战与未来

随着全球数据呈现指数级增长,当前的信息存储技术面临维护成本高昂、存储寿命有限等多个缺陷,逐渐无法满足日益凸显的需求。因此,迫切需要引入新的信息存储方法来解决这一问题。DNA作为一种天然的遗传信息载体,具备高存储密度、潜在低维护成本和长寿命等优势,因此被视为一种有潜力的新型信息存储介质。该文对DNA数据存储技术的基本原理和流程进行了概述,并回顾了其历史发展。同时,对当前基于DNA存储的领域仍面临的挑战进行了总结,如缓慢的数据写入和读取速度等,以及应对这些挑战的一些潜在策略。最后,为了满足全球对新存储方法的需求,该文指出了DNA数据存储技术的未来发展方向。

Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

AIM： To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS： Hpylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction （PCR） and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. co/i DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicib/of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 10^8 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 10^7 CFU of live Hpylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylon 4 wk post-challenge. RESULTS： The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that （100%） in the control group （P 〈 0.01）. CONCLUSION： Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.

Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

Rat calcineurin （CAN） A a isoform （Ppp3ca） cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rht, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG- FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment （3.97 kb） obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect （CPE） appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A a （Ppp3ca） cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylorihpaA

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

Objective:To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines.Methods:BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA;splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro.Results:The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone,but there was not significantly different (P>0.05).Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-γ in supernatant of splenocytes cultured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05).Conclusion:The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

A novel recombinant DNA vaccine encodingMycobacterium tuberculosis ESAT-6 and FL protects againstMycobacterium tuberculosis challenge in mice

Mycobacterium tuberculosis 6-kDa early secretory antigenic target （ESAT-6） is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand （FL） that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid （plRES-epitope-peptides-FL） encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine （prime） and intranasal administration of the epitope peptides （boost） was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Thl cytokines （IFN-γ and IL-12）, the number of IFN-γ＋ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant plRES-epitope-peptides-FL plasmid is a useful DNA vaccine for pre- venting Mycobacterium tuberculosis infection.

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

DNA损伤修复相关通路的合成致死靶点研究及其在卵巢癌中的应用和前景

DNA损伤引发细胞启动一系列DNA损伤应答(DNA damage response,DDR),包括DNA损伤修复、细胞周期检查点激活、细胞周期阻滞、各种细胞内信号转导途径的活化和细胞凋亡等。DNA损伤修复(DNA damage repair)是细胞维持基因组稳定性的重要机制,于2015年获得诺贝尔化学奖。DNA损伤修复途径主要包括:碱基切除修复(base-excision repair,BER)、核苷酸切除修复(nucleotide excision repair,NER)、错配修复(mismatch repair,MMR)、同源重组(homologous recombination,HR)和非同源末端连接(non-homologous end joining,NHEJ)等,分别在DNA单链断裂(single-strand break,SSB)或双链断裂(double-strand break,DSB)等损伤修复中发挥重要作用。DNA损伤修复缺陷与肿瘤发生发展密切相关,同时也是肿瘤治疗的重要靶点。DNA损伤修复通路的多聚ADP核糖聚合酶(poly-ADP-ribose polymerase,PARP)与乳腺癌易感基因BRCA 1/2等存在合成致死(synthetic lethality)作用,使PARP抑制剂(PARP inhibitor,PARPi)成为第一个也是目前唯一上市的肿瘤治疗合成致死靶药。PARPi在卵巢癌及多种实体瘤治疗中疗效良好,使DNA损伤修复及相关DDR通路的合成致死靶药研发成为热点,其他在研靶点主要包括:共济失调毛细血管扩张突变蛋白(ataxia telangiectasia-mutated protein,ATM)、共济失调毛细血管扩张与RAD3相关蛋白(ataxia telangiectasia and Rad3 related protein,ATR)、DNA依赖性蛋白质激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)、细胞周期检测点激酶1(checkpoint kinase1,CHK1)、细胞周期检测点激酶2(checkpoint kinase 2,CHK2)、阻止有丝分裂的蛋白质激酶WEE1等。PARPi与其他DDR靶药、抗血管生成药物及免疫检查点抑制剂的联用,有可能成为克服PARPi耐药、提高疗效的有效手段和发展前景。本文针对DNA损伤修复及相关DDR通路的关键分子和潜在肿瘤治疗靶点进行综述,阐述了DNA损伤修复相关通路的合成致死靶点研究及在卵巢癌的应用和前景,为基础研究及临床应用提供指导。

基于中医证候与精液质量相关参数构建精子DNA碎片预测模型与验证

背景:中医证候与精液质量相关参数相结合,共同预测精子DNA碎片指数(DNA fragmentation index,DFI)异常增高的发生并绘制列线图,能显著提高临床的实操性与应用效能,为临床全面评估精液质量,采取积极干预措施以改善临床结局及制定个体化医疗方案提供依据。目的:探讨基于中医证候与精液质量相关参数构建精子DNA碎片的预测模型与验证。方法:回顾性分析2019年7月至2021年7月在广西壮族自治区南溪山医院中医男科接受中医证候诊断及精子DNA碎片率检查的不育患者共420例,据《人类精液检查与处理实验室手册》(第6版),将其中137例精子DFI>30%患者纳入精子DFI异常增高组,将283例精子DFI≤30%作为对照组;首先采用单因素分析筛选精子DFI异常增高的影响因素,然后采用套索算法(LASSO)校正因子共线性问题并筛选出最佳匹配因子后,将其纳入多因素向前逐步Logistic回归找出其独立影响因素并绘制列线图,最后采用受试者工作曲线、校准曲线、临床决策曲线、临床影响曲线对该预测模型进行区分度与准确度及临床应用效能验证。结果与结论:①单因素分析结果显示,年龄、体质量指数、前向运动率、精子总活率、精子浓度、精子形态学、肾阳虚衰证、湿热下注证、肾精不足证为引发精子DFI异常增高的影响因子(P<0.05);②通过LASSO回归进一步筛选出的最佳匹配因素为年龄、体质量指数、精子总活率、精子浓度、精子形态学、肾阳虚衰证、湿热下注证、肾精不足证(P<0.05);③多因素向前逐步Logistic回归结果显示年龄、体质量指数、精子浓度、精子总活率、湿热下注证、肾阳虚衰证共6项为引发精子DFI异常增高的独立影响因素;④受试者工作曲线显示,模型组曲线下面积为0.760(0.713,0.806),验证组曲线下面积为0.745(0.714,0.776),说明该预测模型具有较好的区分度;⑤校准曲线平均绝对误差0.040,Hosmer-Lemeshow检验P>0.05,表明该模型预测发生精子DFI异常增高的概率与实际发生精子DFI异常增高的概率无显著统计学差异,证实该模型具有较好的准确度;⑥临床决策曲线与临床影响曲线显示,模型组与验证组分别在阈概率值为0.08-0.84与0.09-0.78时具有临床最大净获益,且在该阈概率范围内具有较好的临床应用效能;⑦结果表明,年龄、体质量指数、精子浓度、精子总活率、湿热下注证、肾阳虚衰证为引发精子DFI异常增高的独立影响因素,通过其构建的临床预测模型列线图具有较好的临床预测价值与临床应用效能,可为临床全面评估精液质量、预后与干预及个体化医疗服务提供依据。

不同DNA条形码在微口线虫属形态相近物种分类上的应用

为建立和完善海洋线虫相近物种的DNA条形码鉴定方法,本研究以深圳福田红树林湿地自由生活的海洋线虫优势属微口线虫属(Terschellingia)为研究对象,在形态分类鉴定基础上,将DNA条形码技术引入海洋线虫形态相似物种的鉴定,研究线粒体细胞色素氧化酶第一亚基(COⅠ)基因、18S核糖体RNA基因(18S rDNA)和28S rDNA三种基因序列片段的物种分类效果。研究共鉴定出微口线虫属4个不同的形态学种,获得其中3个种的DNA序列。18S rDNA及28S rDNA两种条形码所构建的发育树支持将本属划分成6个类群;Kimura 2 parameter(K2P)种内和种间阈值分别为18S rDNA的0%~2.5%和0.4%~13.7%,28S rDNA的0%和20.5%~84.6%。18S rDNA的MN18F-Nem_18S_R引物对所扩增的序列最适合作为微口线虫属种类鉴定的DNA条形码,并可用于区分物种复合体。28S rDNA序列虽然能成功扩增,但扩增效率相对较低;COⅠ基因片段无法在所有物种中成功扩增,推测现有引物可能不适合用于本属序列的提取。研究结果表明,DNA条形码可以用于自由生活海洋线虫形态相似物种的识别,但不同的单基因片段对相同物种的鉴定结果有明显差异。

“DNA粗提取和鉴定”实验设计与改进

对DNA粗提取与鉴定实验进行优化,探究植物组织的多种破碎方法对DNA粗提取的影响,并用氯仿等有机物对DNA粗提取物进一步纯化,分析DNA未纯化与纯化后的纯度差异。结果显示,相较于破壁机破碎、加液氮研钵研磨以及玻璃匀浆器研磨,不添加液氮用研钵研磨效果更好。DNA粗提液经纯化后纯度显著提高,蛋白质及盐类等污染明显减少。

利用基于Python/RGB模块的DNA电泳图像分析方法检测绵羊血浆中羊源性成分

该文探究了运用Python处理食品中DNA分子量与含量测定的新方法,建立了DNA凝胶图像分析方法。将不同分子量的DNA Marker与DNA标准样品进行琼脂糖凝胶电泳并拍照,利用自行开发Python程序分析凝胶图,对图像进行灰度图转换、高斯模糊、图像阈值化、轮廓检测的图像优化步骤,探究了轮廓平均值法、轮廓中线法、全局数据平均法、全局数据积分法反映出DNA浓度与RGB数值间的线性关系,选取最优处理方法,通过读取像素迁移距离、RGB-灰度、RGB-向量、RGB-亮度进行DNA分子量与含量的测定实验,建立一种基于Python/RGB色彩体系的DNA凝胶电泳中分子量与含量的分析方法。检测结果误差较小,证明了Python/RGB的DNA分子量与含量分析方法的可行性,同时将该文所构建的凝胶图像分析方法应用于绵羊血浆中羊源性成分检测,结果显示所得目的蛋白为296 bp,而用DNA检测法得出样品中片段大小为294 bp,误差为0.99%,有望构建一种肉类源性成分检测的新方法。

CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

DNA聚合酶θ的合成致死作用研究

利用合成致死(SL)作用治疗癌症可能会成为一种有效且对患者安全的方案。在诱导合成致死效应的众多因素中,参与DNA修复的因素是最密切相关的。当一个经典的DNA双链断裂(DSB)修复途径发生突变时,替代途径可能是消除肿瘤细胞的靶标。目前,抑制缺乏经典修复途径肿瘤细胞中的RAD52和/或PARP1一直是诱导合成致死作用的潜在靶标,但是,常用的PARP1抑制剂(PARPi)的耐药性是制定治疗方案的最大障碍。由POLQ基因编码的DNA聚合酶θ(Polθ)介导的末端连接(TMEJ)在另一种DSB修复途径起关键作用。因此,它是治疗具有同源重组修复(HRR)缺陷肿瘤的潜在靶点,抑制其活性可以诱导SL。在本综述中,主要讨论了基于靶点Polθ合成致死性的抗癌疗法的现状。

Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16 L1/E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.

水生生物环境DNA监测技术的发展、应用与标准化

水生态系统是保障国家生态安全的重要基础。水生生物在水生态系统中扮演着核心角色,是研究水体演变的重要依据,是维护水生态健康的关键。传统水生生物调查和监测通常采用形态学方法,但其存在专业知识要求高、难以标准化和自动化及费时耗力等缺陷。环境DNA(Environmental DNA,简称eDNA)技术是一种通过监测环境中存在的DNA片段来识别特定生物物种的方法,可以实现基于水体中DNA分子进行水生生物的鉴定和监测,为水生生物的常态化监测提供了一个准确、便捷、可标准化和自动化实施的方案。文章介绍了eDNA技术的基本原理,总结回顾了eDNA技术从萌芽到广泛科研应用的发展历史和过程,介绍了基于eDNA的宏条形码和宏基因组等各类水生生物鉴定监测技术;阐述了eDNA技术在保护种、入侵种及生物类群监测和水生态评估等各领域的应用;分析了eDNA技术当前面临的物种参考序列数据库不完善等各类挑战;提出了通过优化完善数据库、样品采集方法、评价指标和参数、样品保藏、数据分析和存储等来推动eDNA技术标准化和自动化,以解决当前面临的挑战。同时,基于eDNA技术当前的发展阶段,提出了在我国水体结合专业形态分类鉴定开展水生生物eDNA技术标准化监测的实施建议。

DNA甲基化对急性髓系白血病作用的研究进展

研究证实,在基因的表观遗传调控中DNA甲基化起着至关重要的作用。而DNA甲基转移酶(DNMT)催化DNA甲基化,这是DNA甲基化模式形成和保持的必要条件。在哺乳动物细胞中,有三种关键的DNMT负责着不同的任务。首先是DNMT1,负责维持DNA的甲基化状态,保持细胞功能正常运转。而另外两种则是DNMT3a和DNMT3b,它们则负责推动DNA从头开始的甲基化过程。目前,急性髓系白血病(AML)的病因仍无法完全阐明。通过研究发现,异常的表观遗传学变化与AML的发病密切相关。深入探讨DNA甲基化与AML之间的联系,将为治疗这种疾病和开发新药物提供关键的分子靶点。这一领域的突破将为医学界带来新的希望,为患者提供更有效的治疗方案。Research has confirmed that DNA methylation plays a crucial role in the epigenetic regulation of genes. DNA methyltransferase (DNMT) catalyzes DNA methylation, which is a necessary condition for the formation and maintenance of DNA methylation patterns. In mammalian cells, there are three key DNMTs responsible for different tasks. Firstly, DNMT1 is responsible for maintaining the methylation status of DNA and ensuring the normal functioning of cells. The other two are DNMT3a and DNMT3b, which are responsible for driving the DNA methylation process from scratch. At present, the etiology of acute myeloid leukemia (AML) cannot be fully elucidated. Through research, it has been found that abnormal epigenetic changes are closely related to the onset of AML. Exploring the relationship between DNA methylation and AML in depth will provide key molecular targets for the treatment of this disease and the development of new drugs. Breakthroughs in this field will bring new hope to the medical community and provide more effective treatment options for patients.