治疗型HBV基因疫苗免疫效果的研究

为探索治疗型HBV基因疫苗治疗慢性乙型肝炎的可行性 ,自行构建了HBV包膜蛋白前S2 ·S基因及人IL 2和IFN γ融合蛋白基因真核表达质粒 ,并将双质粒融合蛋白作为佐剂组合成治疗型HBV基因疫苗 ,在健康小鼠、HBV转基因 (Tg)小鼠、新西兰兔和恒河猴体内进行了免疫效果试验。结果发现 :①特异性CTL活性提高 ;②对HBsAg特异性T细胞增殖能力提高 ,HBsAg 3 0 μg /ml对pcS2 ·S免疫的小鼠脾细胞的刺激指数 (SI =5 6± 0 9)明显较空白质粒pcDNA3 1组 (SI =2 0± 0 5 )为高 (P <0 0 1) ,免疫组的IL 2 /IFN γ分泌水平为 (2 2 6 3± 41 0 5pg/ml) /(5 1 1± 7 7pg/ml) ,明显较空白组的 (69 0± 2 2 1pg/ml) /(0 9± 0 7pg/ml)为高 (P <0 0 1) ;③HBV基因疫苗免疫健康小鼠局部引流淋巴结中DCs的诱导HBsAg致敏T细胞增殖指数 (4 2 0 )较pcDNA3 1组 (2 5 5 )为高 ;⑤用在体电脉冲法注射治疗型HBV基因疫苗后 ,检测血清抗 HBs水平 ,无论是小鼠、兔还是猴均有明显提高。提示该治疗型HBV基因疫苗能较好地诱导体液和细胞免疫 ,为其治疗HBV感染提供了实验依据     

结核病Mtb8.4基因疫苗构建、表达及诱导的细胞免疫应答

目的制备结核病Mtb8.4基因疫苗,并研究其免疫原性。方法克隆结核杆菌新抗原Mtb8.4基因,导入真核表达载体pcDNA3.1( ),构建重组质粒pcDNA3.1( )Mtb8.4,转染COS7细胞后,用RTPCR检测Mtb8.4基因在细胞内正确表达。将Mtb8.4基因疫苗肌肉注射免疫C57BL/6N小鼠,第3次DNA免疫后4周,处死小鼠,制备免疫小鼠脾细胞,脾细胞培养上清检测细胞因子水平;并按效、靶比例分别为100∶1、50∶1、10∶1进行CTL杀伤检测。结果Mtb8.4基因疫苗组免疫小鼠脾细胞培养上清中γ干扰素(IFNγ)和白细胞介素2(IL2)含量分别为787.317±45.586pg/ml和319.953±57.978pg/ml,BCG组分别为1486.540±39.600pg/ml和767.043±50.269pg/ml,BCG组IL4的含量为90.580±10.998pg/ml,明显高于其他各组(P<0.01)。效靶比为100∶1、50∶1、10∶1时,Mtb8.4基因疫苗组的CTL活性分别为55.3%、35.7%、9.2%,BCG组分别为28.9%、21.4%、9.8%。Mtb8.4基因疫苗主要使抗原特异性Th1型细胞免疫应答增强,细胞因子IFNγ和IL2分泌增加,IL4分泌减少,CTL活性增加;而BCG使IFNγ、IL2和IL4分泌均增加。结论结核病Mtb8.4基因疫苗能诱导较强的Th1型细胞免疫应答,可作为结核病的候选疫苗。     

大剂量HBsAg疫苗对HBV转基因小鼠细胞免疫调节的研究

目的　研究大剂量HBsAg疫苗对HBV转基因小鼠 (Tg鼠 )细胞免疫应答的影响。方法　Tg鼠 4 8只 ,每只注射 6 μg(相当于人产生抗体剂量的 180倍 )的HBsAg疫苗后 ,用流式细胞术、氚 胸腺嘧啶核苷 (3 H TdR)掺入法和ELISA法 ,分别检测其树突状细胞(DC)、T淋巴细胞增殖及其诱生细胞因子IL 2、IFN γ的水平。结果　大剂量HBsAg疫苗组DC表面共刺激分子CD80、CD86和I Ek的阳性百分率 (% )均高于对照组 (P <0 0 5 ) ,特异T淋巴细胞增殖能力 8个月的cpm值 (10 0 77 2± 85 74 0 )显著高于 1周的 (5 32 9 1± 30 86 0 ) (P <0 0 5 ) ;其诱生细胞因子的水平 8个月时IL 2 (46 2 4± 12 2 5pg/ml)、IFN γ(976 1± 5 4 4 1pg/ml)显著高于 1周时IL 2 (15 6 1±78 7)、IFN γ(5 8 3± 4 9 5 ) (P <0 0 1)。结论　大剂量HBsAg疫苗能够增强Tg鼠细胞免疫应答能力 ,恢复对乙型肝炎抗原的应答。     

增强子Ⅰ对乙型肝炎病毒DNA疫苗免疫应答的影响

目的 研究乙型肝炎病毒（HBV）自身增强子I（enhancerI，ENHI）对HBV DNA疫苗免疫应答的影响。方法 采用PCR法以HBVadr亚型全基因DNA序列为模板分别扩增表面抗原（HBsAg）和HBsA-ENHI基因片段，重组到载体VR1012中，构建两种HBV DNA疫苗，转染CADS-7细胞及HepG2细胞并免疫BALB／c小鼠。采用蛋白印迹、ELISA、ELISPOT等方法检测其在COS-7和HepG2细胞内的表达及小鼠的体液及细胞免疫应答效果。结果 转染的HepG2和COS-7细胞均表达HBsAg;连接ENHI的HBV DNA疫苗转染HepG2细胞后HBsAg表达量明显升高，两种疫苗转染COS-7细胞表达HBsAg无明显差异；免疫小鼠后第2周产生HBsAb及HBsAg特异性细胞毒T淋巴细胞（CTL），两种疫苗免疫产生的HBsAb及HBsAg特异性CTL无明显差异。结论ENHI可使HBV DNA疫苗转染HepG2细胞表达HBsAg明显增加，对转染COS-7细胞表达HBsAg及接种BALB／C小鼠引起的免疫应答无明显影响。     

热休克蛋白65增强小鼠对HBV DNA 疫苗免疫反应的研究

目的:观察热休克蛋白65的真核表达载体(pHSP65)对HBV DNA疫苗诱导BALB/c小鼠(H-2d)免疫应答的调节作用。方法:肌内注射空载体pcDNA3,HBV DNA疫苗加HSP65佐剂(pHBVS2S pHSP65)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs抗体;ELISPOT检测分泌IFN-γ的脾淋巴细胞;4 h51Cr释放法检测小鼠脾细胞CTLs活性。结果:HBV DNA佐剂组免疫小鼠抗HBsAg抗体滴度虽高于不加佐剂组,但无显著性差异;其IgG1/IgG2 a的比例不同于多肽免疫组,二者分别为0.395与10。佐剂组小鼠分泌IFN-γ的脾淋巴细胞量是不加佐剂组的2~3倍。CTLs细胞杀伤活性(E∶T=100)佐剂组与不加佐剂组分别为:(53.68±7.5)%、(42.81±7.7)%,差异显著(P<0.05)。结论:HBV DNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTLs反应;HSP65佐剂能够有效提高小鼠对DNA疫苗的细胞免疫应答,有望成为DNA疫苗的免疫佐剂。     

电脉冲肌注法增强治疗型HBV DNA疫苗免疫效果的实验研究

为提高细胞内质粒DNA的导入率并增强DNA疫苗诱导的免疫效果 ,采用在体电脉冲肌注法接种治疗型HBVDNA疫苗。结果接受电脉冲(2 0 0V/cm)肌注的 8只BALB/c小鼠局部肌肉组织荧光素酶活性为 1 6 1 70± 1 2 5 33RLU ,未加电脉冲对照组为 8 0 2±8 0 0RLU ,相差 4个数量级 ,差异具非常显著性 (P <0 0 0 5 ,t=3 6 74 )。新西兰兔电脉冲肌注法接种后第 2、4周 ,双质粒 (pS2 ·S pFP)大剂量组 (5 0 5 0 μg/只 )血清抗 HBs阳性动物数明显较同期未加电脉冲对照组 (大剂量 :1 0 0 0 1 0 0 0 μg/只 )高 ,差异具显著性意义 (P <0 0 5 ) ;第 1 3周时 ,电脉冲免疫大 (5 0 5 0 μg/只 )、中 (2 5 2 5 μg/只 )、小 (5 5 μg/只 )剂量组均有血清抗 HBs阳性鼠出现 ,分别为 5、4和 4 只 ,组间阳性动物数并无显著性差异 (P >0 0 5 ) ,但均较同期的对照组高 ,并具有显著性意义 (P <0 0 5 )。恒河猴电脉冲肌注法接种后 8周血清抗 HBs阳性动物数为大剂量 (1 0 0 0 1 0 0 0 μg/只)组 3/ 3只 ,中剂量 (5 0 0 5 0 0 μg/只 )组 2 / 3只 ,小剂量 (1 0 0 1 0 0 μg/只 )组 0 / 3只 ,大剂量组与小剂量组及非电脉冲对照组之间比较差异具有显著性意义 (P <0 0 5 ) ;至免疫第 1 3周时 ,电脉冲免疫的大、中、小 3个剂量组     

结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12表达质粒联合免疫的细胞免疫应答观察

目的 观察结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12(hIL-12)联合免疫小鼠所诱导的细胞免疫应答.方法 15只C57BL/6N小鼠随机分为Mtb8.4基因疫苗 hIL-12质粒组(联合免疫组)、Mtb8.4基因疫苗组、卡介苗(BCG)组、空载体组和PBS组,基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106 CFU BCG免疫1次.ELISA法检测小鼠脾细胞培养上清中细胞因子水平;乳酸脱氢酶(LDH)释放法检测免疫小鼠特异性细胞毒性T淋巴细胞(CTL)杀伤活性.结果 联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-γ和IL-2水平(分别为1 493.340±8.128pg/ml、747.489±48.676pg/ml)显著高于Mtb8.4基因疫苗组,与BCG组相当,IL-4分泌减少,特异性CTL杀伤活性增强.结论 hIL-12表达质粒能够增强Mtb8.4基因疫苗所诱导的细胞免疫应答.     

泛素-结核抗原融合基因DNA疫苗诱导的小鼠细胞免疫应答

目的 构建结核杆菌Ag85A抗原DNA疫苗(pA)和泛素基因与Ag85A抗原基因融合的DNA疫苗(pUA),观察疫苗在小鼠中的免疫应答.方法 将构建的DNA疫苗pA和pUA分别肌肉注射免疫BALB/c雌性小鼠,检测小鼠的血清抗体(IgG、IgG1、IgG2a)、细胞因子(IFN-γ、IL-4)和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗和单基因DNA疫苗诱导的免疫应答的强度.结果 pA组小鼠血清IgG水平高于pUA组(P＜0.01),但IgG2a/IgG1比值(4.16±0.20)低于pUA组(7.88±0.30)(P＜0.01).与pA组比较,pUA组小鼠IFN-γ分泌水平增高(P＜0.01),IL-4分泌水平下降(P＜0.01);pUA组的CTL活性高于pA组.结论 融合的DNA疫苗pUA诱生的抗原特异的体液免疫应答不及单基因DNA疫苗pA,但其能诱导更强的细胞免疫应答.提示泛素-Ag85A融合基因DNA疫苗对于防治结核病比单基因DNA疫苗更为有效.     

增强子Ⅰ对乙型肝炎病毒DNA疫苗免疫应答的影响

目的研究乙型肝炎病毒(HBV)自身增强子Ⅰ(enhancerⅠ,ENHⅠ)对HBVDNA疫苗免疫应答的影响。方法采用PCR法以HBVadr亚型全基因DNA序列为模板分别扩增表面抗原(HBsAg)和HBsAg-ENHⅠ基因片段,重组到载体VR1012中,构建两种HBVDNA疫苗,转染COS-7细胞及HepG2细胞并免疫BALB/c小鼠。采用蛋白印迹、ELISA、ELISPOT等方法检测其在COS-7和HepG2细胞内的表达及小鼠的体液及细胞免疫应答效果。结果转染的HepG2和COS-7细胞均表达HBsAg;连接ENHⅠ的HBVDNA疫苗转染HepG2细胞后HBsAg表达量明显升高,两种疫苗转染COS-7细胞表达HBsAg无明显差异;免疫小鼠后第2周产生HBsAb及HBsAg特异性细胞毒T淋巴细胞(CTL),两种疫苗免疫产生的HBsAb及HBsAg特异性CTL无明显差异。结论ENHⅠ可使HBVDNA疫苗转染HepG2细胞表达HBsAg明显增加,对转染COS-7细胞表达HBsAg及接种BALB/c小鼠引起的免疫应答无明显影响。     

治疗型双质粒HBV DNA疫苗的构建及其鉴定

为构建以人白细胞介素 2 /γ干扰素(hIL 2 /hIFN γ)融合基因为佐剂的治疗型HBVDNA疫苗 ,采用DNA重组技术分别构建含有HBV包膜中蛋白 (preS2 ·S)抗原和hIL 2 /hIFN γ融合蛋白的真核表达质粒即pcDNAS2 ·S和pcDNAIIF ,酶谱和测序分析表明克隆的preS2 ·S和hIL 2 /hIFN γ融合蛋白基因片段的方向、序列与预期相符。用脂质体转染试剂转染COS 7细胞 ,并用ELISA检测转染细胞培养上清中目的基因表达水平。结果显示 ,质粒转染后 4 8h达峰值 ,分别为HBsAg(P/N) =7.6 3、IL 2 =1 0 .35ng/ml、IFN γ =7.90ng/ml。以CTLL 2依赖细胞株/MTT比色法和微量细胞病变抑制法 (WISH VSV)检测pcDNAIIF转染后 4 8h培养上清中IL 2及IFN γ活性 ,结果分别为 998U/ml和 2 4 9U/ml。证明构建的重组质粒pcDNAS2 ·S和pcDNAIIF结构正确 ,在体外转染细胞其质粒得到分泌表达并保留生物学活性     

Methods enhancement for improved recovery of human DNA from forensic blood samples on different fabrics using the DNA IQ System

DNA typing of biological samples can be a challenging task due to the presence of small amounts of DNA and/or a high content of PCR inhibitors. Our aim is to develop a protocol to recover DNA suitable for DNA typing from blood-stained fabrics based on the DNA IQ System. Blood stains on different fabrics were buried in different types of soil and left for 1–7 days. The samples were then recovered and DNA was extracted with a modified DNA IQ System protocol, involving a modification in the preparation of the Wash Buffer. The samples extracted with the modified protocol showed a higher amount of recovered DNA compared with the recommended protocol. The removal of isopropanol from the Wash Buffer composition contributes to a higher amount of recovered DNA.     

The origin of unknown source DNA from touched objects

The presence of DNA in a criminal investigation often requires scrutiny in relation to how it came to be where it was found. There is a paucity of data with respect to the extent to which one can assume that the last person handling an object, which has previously been touched by others, will contribute to the DNA profile generated from it. There are limited data in detailing the extent to which any foreign DNA is picked-up from a previously touched object and transferred to subsequently touched objects. This study focuses on DNA transfer and persistence on a knife handle after multiple handlings with the knife by different individuals soon after each other, as well as handprints left on flat DNA-free surfaces immediately after touching a knife handle with a known history of prior handling.The profiles of later handlers of a knife are more prominent than earlier handlers; however, the last handler is not always the major contributor to the profile. Proportional contributions to the profiles retrieved from knife handles vary depending on the individuals touching the knife handle. They can also vary when knife handles have been handled in the same manner by the same individuals in the same sequence on different occasions. Hands readily pickup DNA left on objects by others and transfer it to subsequently touched objects. The quantity of foreign DNA picked up by a hand and deposited on subsequently touched objects diminishes as more DNA-free objects are handled soon after each other.Caution is advised when considering how DNA from different individuals may have been transferred to the object from which it was collected.     

Radiation-induced DNA Damage and Its Repair

Summary

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes.

A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment ³²P-labelled at one end, it has been shown that the alkali—labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

Are we fishing or catching? Evaluating the efficiency of bait capture of CODIS fragments

This study sought to document the efficiency of DNA bait capture (i.e., “fishing”) methods by two measures: (1) its ability to retain targeted DNA molecules, and (2) its ability to remove non-target DNA molecules from a pool containing both. DNA bait capture uses synthetic biotinylated DNA primers to bind target DNA, which are then immobilized onto streptavidin coated magnetic beads and drawn to a magnet. Bound DNA should, therefore, be isolated from non-target DNA and impurities (e.g., PCR inhibitors) and can be later eluted from the beads for downstream applications. Efficiencies were estimated by comparing the number of “copies in” to “copies out” with quantitative polymerase chain reaction (qPCR). Retention of target DNA molecules, ranging from 109 to 288 base pairs (bps) in length, averaged just 9.06–3.53% (i.e., loss of 90.94–96.47%) using the fishing protocol as originally described. Some improvement was achieved by employing a modified protocol (i.e., with a shortened hybridization time, use of twice the amount of M-270 streptavidin-coated beads, and modified bead washing), resulting in average retention of 31.41–12.08% of the same set of targeted molecules. Noted was the lack of efficacy in removing non-target DNA molecules as opposed to targeted molecules. It was also observed that most of the molecules (61.35–69.49%) are “lost” during the essential hybridization step of the fishing protocol, suggesting its suitability for high copy number samples only. While the bait capture method may be useful in the study of polymerase chain reaction (PCR) inhibited DNA samples as previously suggested, it is necessary to carefully weigh this possible advantage against the degree of expected DNA loss and the non-selectivity of the method for targeted over non-targeted DNA.     

DNA within cars: prevalence of DNA from driver,passenger and others on steering wheels

ABSTRACT

Cars are frequently involved in criminal activities and sampled for DNA to assist investigations. To improve our awareness of DNA transfer, persistence, prevalence and recovery (DNA-TPPR) within cars we studied DNA profiles from samples collected from several sites in the front compartment of cars with known histories and occupancies. Here findings relating to steering wheels are reported. Each of the four quarters of the rim as well as the centre column, of four cars, provided good quantities of DNA for profiling. The driver was observed as the sole, major or co-major in 19/20 profiles, and as a minor in the remaining profile generated from these samples. Known close associates, including co-resident partners and passengers/friends, as well as other unknown individuals, who had not driven the car, are also detected on many of the sampled steering wheel sites. More studies are required to improve our awareness of DNA-TPPR and to generate data to help determine probabilities for different profile types and levels of specific contributions given specific circumstances relating to steering wheels, as well as several other relevant areas within cars, to assist sample targeting and activity level assessments.     

What’s on the bag? The DNA composition of evidence bags pre- and post-exhibit examination

Current forensic DNA profiling kits and techniques enable the detection of trace amounts of DNA. With advancements in kit sensitivity, there is an increased probability of detecting DNA from contamination. Research into DNA transfer within operational forensic laboratories provides insight into the possible mechanisms that may lead to exhibit contamination. To gain a greater understanding of the potential for evidence bags to act as DNA transfer vectors, the level of DNA accumulating on the exterior of evidence bags during the exhibit examination process was investigated. The exterior of 60 evidence bags were tapelifted before and after the examination of the exhibit inside of the bag resulting in 120 DNA profiles. These DNA profiles were compared to DNA profiles of staff working within the building and samples taken from the exhibit inside the bag. Common DNA profile contributors from each sample were also identified through STRmix™ mixture to mixture analysis. The average DNA quantity and number of profile contributors was higher in samples taken from the bag before exhibit examination than after examination. Fifty six percent of all samples taken identified a match between DNA recovered from the evidence bag and at least one staff member. On 11 bags, a common contributor was identified between the exhibit in the bag and the exhibit package post-examination. In one instance a DNA profile, matching that of a donor, on the exhibit bag before examination was also detected on a sample taken from the exhibit, raising the possibility of outer bag-to-exhibit DNA contamination. This study demonstrates that operational forensic laboratories must consider exhibit packages as a potential source of DNA contamination and evaluate their exhibit handling and storage procedures accordingly.     

Differential reporting of mixed DNA profiles and its impact on jurists’ evaluation of evidence. An international analysis

While DNA analysis is considered by many the gold standard in forensic science, there is ample room for variation in interpretation and reporting. This seems especially the case when working with (complex) mixed DNA profiles. Two consecutive studies on differential DNA reporting were conducted. In Study 1, we first examined type and magnitude of differences when forensic DNA experts across institutes and jurisdictions are handed an identical forensic case with mixed profiles. In Study 2, we explore the impact of the observed differential reporting on jurists’ evaluation of the DNA evidence.19 DNA expert reports from forensic institutes across Western jurisdictions were obtained. Differences between the reports were many and include extensiveness of the reports, explanations of technical issues, use of explanatory appendices, level of reporting, use of context information, and, most markedly, type and substantive content of the conclusions. In Study 2, a group of criminal law students judged a selection of these reports in a quasi experimental study design. Findings show that these differing reports have quite different evidentiary value for jurists, depending on which expert authored the report. It is argued that the impact of differential reporting on jurists’ evaluation was so fundamental and substantive that it is seems reasonable to claim that in an actual court case it could make the difference between acquittal and conviction.     

DNA recovery from fired hollow point ammunition

ABSTRACT

Firearm-related exhibits are often found at crime scenes. These exhibits may include the firearm, cartridges, cartridges cases or bullets. As ammunition needs to be handled to load the weapon, regardless of the action or loading type, DNA may be deposited onto the ammunition via touch. As reproducible DNA profiles have been obtained from fired cartridge casings and Improvised Explosive Device (IED) fragments, it is possible that quantifiable amounts of DNA could be recovered from fired bullets. A series of 40 Winchester PowerPoint 22LR 42 grain HP Copper Plated bullets were loaded with serially diluted cell suspensions obtained from a female donor. These were shot into 500 sheet reams of A3 paper for capture and returned to a sterile DNA laboratory for removal, extraction and quantification of DNA. Repeatable partial profiles with five reportable loci pairs consistent with the cell donor were obtained in one replicate of the neat sample. Weak partial profiles were also present in 1:2, 1:5 and 1:10 dilutions. To our knowledge, this is the first reported evidence of DNA surviving the cycle of fire and being recovered from a fired bullet under controlled conditions.