单克隆非特异性抑制因子—β（MNSFβ）在大肠杆菌中的表达、抗体的制备及在小鼠子宫内膜上的组织定位

为了获得足够量的单克隆非特异性抑制因子-β蛋白（monoclonal nonspecific supressor factorβ,MNSFβ）及其抗体用于探讨MNSFβ在着床中的作用机理,本研究构建了表达质粒pBV220/MNSFβ-hCCβ,在大肠杆菌中表达了融合蛋白MNSFβ-hCCβ,用抗hCGβ抗体对表达产物进行鉴定,结果表明融合蛋白MNSFβ-hCGβ得到了正确表达,且分子量和理论值相近,表达产物MNSFβ-hCGβ经初步纯化后用于免疫Balb/C小鼠,制备抗体,同时,我们还构建了表达质粒pGEX-4T-2/MNSFβ,在大肠杆菌中表达了融合蛋白GST-MNSFβ,用融合蛋白GST-MNSFβ对融合前的免疫小鼠回忆刺激并进行检测,制备了抗MNSFβ多克隆抗体和单克隆抗体,应用所制备的多克隆抗体进行免疫组化研究,对MNSFβ在小鼠子宫内膜上进行了组织定位,结果显示,MNSFβ在着床日（受精后第4.5天）小鼠子宫非着床位点的表达和着床位点相比明显提高。     

小鼠睾丸特异性基因Vad1.2重组蛋白的构建及多克隆抗体制备

目的：构建小鼠睾丸特异性基因Vad1.2重组蛋白,制备多克隆抗体。方法：将小鼠睾丸组织Vad1.2转录本行RT-PCR扩增,通过DNA重组技术插入克隆载体p ET15b,进行酶切及DNA序列分析。将表达载体转化入大肠杆菌BL21（DE3）RIL感受态细胞,10 mmol/L IPTG诱导表达重组蛋白,通过10%SDS-PAGE、Western blotting及质谱分析进行鉴定。随后对重组Vad1.2蛋白进行纯化和进一步鉴定。最后,制备兔抗小鼠Vad1.2多克隆抗体,并通过Vad1.2-EGFP质粒转染GC-2spd（ts）细胞对其特异性进行验证。结果：大肠杆菌BL21（DE3）RIL细胞中诱导表达并纯化的小鼠Vad1.2重组蛋白构建正确并经Western blotting和质谱分析得到证实。所制备的多克隆抗体可特异性识别GC-2spd（ts）细胞中过表达的Vad1.2-EGFP融合蛋白。结论：成功构建小鼠Vad1.2重组蛋白,并制备多克隆抗体,为Vad1.2基因的进一步研究奠定了实验基础。     

转基因小鼠过表达人Lin28A/Lin28B对小鼠体重的影响

目的:Lin28是一种高度保守的RNA结合蛋白,对生物体的生命活动具有重要的调节作用.为了研究人Lin28A与Lin28B基因的功能,我们构建了分别过表达人Lin28A或Lin28B基因的两种转基因小鼠,表型分析发现小鼠体重变化,为研究Lin28A和Lin28B基因的生物学功能提供模型动物.方法:分别将人类Lin28A与Lin28B cDNA插入PCAG启动子下游,构建Lin28A与Lin28B转基因表达载体,通过显微注射方法,分别建立Lin28A转基因小鼠与Lin28B转基因小鼠.PCR鉴定转基因小鼠的基因型,筛选出人类Lin28A与Lin28B表达的小鼠,统计两种转基因小鼠体重变化.结果:①经PCR鉴定与公司测序证明Lin28A和Lin28B两种载体构建成功.②PCR鉴定小鼠基因型,筛选出可以特异性表达人Lin28A或Lin28B的转基因小鼠,并比较转基因小鼠与同窝野生型小鼠体重,发现两种转基因小鼠体重均大于同窝野生型小鼠,说明在小鼠体内过表达Lin28A或Lin28B均能引起小鼠体重增加.结论:人Lin28A与Lin28B在转基因小鼠体内能正常表达,并且能发挥生物学功能.又因为Lin28A和Lin28B在小鼠体内过表达会引起小鼠体重增加,我们推测其可能通过影响小鼠糖代谢过程引起小鼠体重改变.构建的Lin28A和Lin28B的转基因小鼠为进一步研究Lin28A和Lin28B在人新陈代谢、生长和发育过程中的作用提供了动物模型.     

抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器的制备与验证

目的:构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体并制备和验证抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型。方法:利用PCR法扩增出抗人p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L,然后分别将嵌合抗体重链基因H和嵌合抗体轻链基因L连接到乳腺特异性表达质粒pBC1,从而构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L。分别将抗p185~(erbB2)人鼠嵌合抗体ChAb26乳腺特异表达载体pBC1-H和pBC1-L线性化,然后使用原核显微共注射法获得8只转基因FVB小鼠,通过鼠尾直接PCR鉴定其转基因阳性。通过RT-PCR、荧光定量PCR鉴定转基因小鼠乳腺组织中抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达。使用小鼠乳汁采集器收集其乳汁并通过Western blot和夹心ELISA等实验鉴定抗p185~(erbB2)人鼠嵌合抗体ChAb26是否获得表达。结果:经测序验证,抗p185~(erbB2)人鼠嵌合抗体ChAb26的嵌合重链基因H和嵌合轻链基因L分别与乳腺特异表达质粒pBC1正确正向连接。鼠尾直接PCR结果显示所获8只转基因FVB小鼠均为转基因双阳性小鼠,且抗p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L在它们的后代中稳定遗传,它们的后代中转基因小鼠双阳性率约为30%; RT-PCR和荧光定量PCR的结果显示,转基因双阳性小鼠及其双阳性后代的乳腺组织中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达; Western blot和ELISA等实验结果显示,转基因双阳性小鼠乳汁中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的蛋白质表达,而且抗p185~(erbB2)人鼠嵌合抗体ChAb26与羊抗人κ链抗体和羊抗人Ig G Fc-HRP抗体均能特异性结合。结论:成功构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L和制备了抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型,为今后抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因牛乳腺生物反应器的研究奠定了理论和技术基础。     

同种异体淋巴细胞致狼疮肾炎小鼠模型的研究

目的　制备狼疮肾炎小鼠模型。方法　采用昆明小鼠同种异体脾淋巴细胞经Lectin刺激后输入小鼠腹腔皮下的方法。结果　成功地制备了系膜增生型狼疮肾炎小鼠模型 ,通过光镜、免疫荧光、电镜观察其病理形态、并检测了血清抗ds -DNA抗体、补体以及尿蛋白 ,证实了该模型的可靠性。结论　制备的系膜增生型狼疮肾炎小鼠模型有实用价值     

前列腺素受体HA标签小鼠的鉴定

前列腺素是一类有重要生理活性的不饱和脂肪酸衍生物,其通过结合特异性的受体发挥生理功能。前列腺素受体缺乏特异性的抗体,这极大制约着前列腺素信号直接的深入解析。本文旨在鉴定及验证"人造精子"和CRISPR-Cas9联合应用构建的氨基端(-NH2,-N)带HA标签的9种前列腺素受体小鼠。通过将向导RNA表达质粒和带标签的打靶载体质粒转入"人造精子细胞",筛选出含HA标签的"人造精子细胞"。进一步将标签细胞注射到小鼠卵母细胞中并移植入假孕母鼠体内,获得标签小鼠。提取前列腺素受体HA标签小鼠的基因组DNA,利用PCR技术检测小鼠的基因型。分离小鼠腹腔巨噬细胞,通过Western blot方法验证带HA标签的前列腺素受体的蛋白表达情况。结果显示,前列腺素受体HA标签小鼠能扩增出特异性的DNA条带,并且巨噬细胞蛋白中可检测出特定HA蛋白条带的表达,野生型小鼠则没有相应条带。综上可知,我们成功制备了9种前列腺素受体-N-HA标签小鼠,为进一步研究前列腺素信号在体内及体外的病理生理功能提供强有力的工具。     

幽门螺杆菌致人胃病的小鼠模型研究

目的建立幽门螺杆菌致人胃病的小鼠模型.方法用Ⅰ型幽门螺杆菌(Helicobacter pylori,Hp)对三种SPF级小鼠(BALB/c、NIH、 KM)采用循环滴喂攻击等方法处理,从菌体定值、抗体水平、病理变化三个方面分14、39、69、105 d进行测试.结果 Hp可持续定植于各供试小鼠胃粘膜上,并刺激Hp抗体(IgG)维持一较高水平,Hp在小鼠上主要引起以胃粘膜变性坏死为主,伴有淋巴细胞浸润的炎症反应,这为Hp致人胃病的症状相似.结论为Hp的致病机理,治疗药物和疫苗研究提供了良好的小动物模型.     

狼疮样肾炎小鼠模型研究进展

系统性红斑狼疮（ systemic lupus erythematosus, SLE）是一种以体内产生抗核抗体为特征的自身免疫性疾病,其病因目前未明,一般认为是遗传、环境、内分泌、感染等因素相互作用的结果。狼疮样肾炎小鼠模型与人SLE发病机制和病理变化等有着相似特点,是研究SLE、筛选抗炎免疫药物较理想的实验性病理模型。本文对近年来狼疮样肾炎小鼠模型制备的方法及评价进行了综述。     

小鼠子宫珠蛋白结合蛋白的生物信息学分析及其多克隆抗体制备

本文旨在制备小鼠子宫珠蛋白结合蛋白(mouse uteroglo binbinding protein,mUGBP)多克隆抗体,为后续研究工作奠定基础。通过生物信息学分析方法预测mUGBP蛋白的跨膜结构、理化特性、疏水性等因素,设计出两段多肽,分别与匙孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)交联后免疫新西兰兔,结果显示其中一个含13个氨基酸残基的多肽序列(221st～233rd)可用作抗原免疫动物,并成功获得高效价的抗mUGBP多克隆抗体。通过ELISA法检测其效价为1:108,亲和层析纯化后Westernblot检测抗体特异性,并将制备的抗体应用于免疫印迹及人和小鼠肺组织免疫组织化学和免疫荧光检测,结果显示本研究制备的抗体具有特异性,且用该抗体成功在人和小鼠气道上皮细胞和肺血管内皮细胞检测到UGBP蛋白表达。结果表明,本研究通过生物信息学方法成功预测了抗原表位,并据此预测成功制备了高效价、高特异性的抗mUGBP多克隆抗体。     

转基因小鼠的研究

编码人生长激素的结构基因(hGH)与编码小鼠金属硫蛋白的基因启动子(MT一1)融合,用显微注射法将此融台基因导入小鼠受精卵的原核中,共注射了121个受精卵、将卵移入11只假孕母鼠的输卵管中,11只母鼠中的7只生了43只小鼠。在小鼠的饮水中加入锌(zn。+)可诱导基因表达。小鼠长到30天时开始称体重、每隔10天记录体重、并与同时出生的对照小鼠体重比较。第一代雄性小鼠体重较对照组重、统计学分析有明显差异。小鼠长到三个月,切尾制备DNA,DNA斑点杂交法和Southern杂交法检测基因整合情况,43只小鼠中有18只有融合基因整合。用Northern、杂交法检测转录的人生长激素mRNA。用这三种方法同样检测了第二代和第三代小鼠,发现融合基因能代代相传,而后代小鼠的表型只在个别小鼠中保留下来。绝大多数转基因小鼠后代的体重减轻。用带人生长激素基因或不带人生长激素基因的小鼠做双亲,进行不同组合的交配,所得到的后代也不相同。     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.     

Hyperexpression of N-acetylglucosaminyltransferase-III in liver tissues of transgenic mice causes fatty body and obesity through severe accumulation of Apo A-I and Apo B

N-Acetylglucosaminyltransferase (GnT)-III catalyzes the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in beta(1-4) configuration in the region of N-glycans and forms a bisecting GlcNAc. To investigate the pathophysiological role of dysregulated glycosylation mediated by aberrantly expressed GnT-III, we generated transgenic mice hyperexpressing the human GnT-III in the liver by introducing human GnT-III cDNA under the control of mouse albumin enhancer/promoter. Total five transgenic founder mice (pGnTSVTpA-10, -14, -20, -25, and -51) expressed the human GnT-III in their livers and were characterized by molecular genetic means. The copy number of transgene integrated into the genome of these mice ranged between 1 and 3 copies per haploid genome. Northern and Western blot analyses showed that the transgene is specifically expressed in the liver but not in any other tissues tested. The triglyceride level in GnT-III transgenic mice was significantly decreased, however, no significant differences in the levels of glucose, cholesterol, or albumin were observed between transgenic and nontransgenic mice. Although glutamate oxaloacetic transaminase and glutamic pyruvic transaminase activities of transgenic mice were also higher than those of nontransgenic mice, no differences in total bililubin and total protein were observed between the two animal lines. Large amounts of apolipoprotein (Apo) A-I and Apo B were specifically detected in the intracellular liver of transgenic mice. The accumulation of Apo A-I in hepatocytes may be due to aberrant glycosylation, since glycosylated Apo A-I was not observed in transgenic mice. However, the accumulated Apo B was severely glycosylated. Therefore, it is suggested that highly expressed transgenic GnT-III allowed unknown target proteins to be glycosylated in large amounts, and the resulting target protein(s) disrupted in assembly formation of Apo A-I in the hepatocytes and cause a decrease in the release of lipoproteins and accumulations of Apo A-I and Apo B in the liver. The transgenic mice showed aberrant glycosylation by GnT-III, resulting in numerous lipid droplets in liver tissues and the obesity. These mice showed microvesicular fatty changes with abnormal lipid accumulation in the hepatocytes. Our study provides the basis for future analysis of the role of glycosylation in hepatic pathogenesis. In the transgenic mice, Apo A-I and Apo B were significantly increased compared with levels in nontransgenic liver tissues.     

Skin-specifically transgenic expression of biologically active human cytoxic T-lymphocyte associated Antigen4-Immunoglobulin (hCTLA4Ig) in mice using lentiviral vector

Xenogeneic skin, especially porcine skin, has already been used to cover large wounds in clinic practice of wound care. Our previous data showed that transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic burn wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong its survival in xenogeneic wounds for coverage. Lentiviral transgenesis provides an extremely efficient and cost-effective method to produce transgenic animals. However, tissue-targeted transgenic expression of biologically functional protein by lentiviral transgenesis is rarely reported. In this work, a recombinant lentiviral vector (LV), named FKCW in this article, was constructed by inserting a skin-specific hCTLA4Ig expression cassette consisting of keratin 14 (K14) promoter, hCTLA4Ig coding sequence and an intronic fragment. Its efficacy for transgenesis and skin-specific expression of bio-active hCTLA4Ig protein was tested using mice as models. The LV FKCW was readily to be packaged and concentrated to high titres (1.287-6.254 × 10(9) TU/ml) by conventional lentivirus package system. Using eggs collected from only five mated females having been subjected to conventional super-ovulation treatment, 8 hCTLA4Ig transgenic founder mice were generated with the concentrated FKCW vector, and transgenic founder per injected and transferred egg was 6.3%, which was nearly 9-fold higher than that for DNA micro-injection with a similar transgene construct in our previous work. The lentiviral transgenic hCTLA4Ig exhibited strictly skin-specific expression at a level comparable to or even slightly higher than that of transgenic hCTLA4Ig delivered by micro-injection in a similar cassette. Lentiviral transgenic hCTLA4Ig protein remarkably suppressed human lymphocyte proliferation in vitro to a degree comparable to that of commercially purchased purified hCTLA4Ig protein with defined activity at similar concentrations. Besides, lentiviral hCTLA4Ig transgenic mouse skin grafted into rat burn wounds exhibited remarkably extended survival compared to wild-type skin of the same strain (13.8 ± 3.8 vs. 6.8 ± 3.0 days), indicating that lentiviral transgenic hCTLA4Ig did inhibit immune rejection against xenogeneic skin graft in vivo. These results laid down the foundation to further efficiently generate transgenic pigs skin-specifically expressing bio-active hCTLA4Ig by lentiviral transgenesis, and provided a demonstration that transgenic animals with tissue-targeted expression of biologically functional protein can be efficiently produced using LV.     

转基因小鼠乳腺表达G-CSF的研究

用PCR法从正常中国人脐带血提取总DNA作为模板,扩增出1.5 kb的人G-CSF基因组基因。序列分析证实其正确性。将其插入小鼠乳清酸蛋白(WAP)基因的起始密码子ATG前的KpnⅠ位点,使其受控于2.6kb的WAP调控序列,构建成乳腺表达载体pWGG。回收经EcoRⅠ酶切后的8.7kb片段用于显微注射。共注射1200枚受精卵,移植34受体母鼠,产仔鼠85只。经PCR检测和DNA印迹分析,证实获得两只整合有人G-CSF基因的雄性鼠,整合率为2.37％。建立的转基因鼠系表明,采用ELASA方法对F1代雌鼠乳汁检测,成功地表达出人G-CSF。表达量为120～250ng/ml。这一结果表明转基因的表达具有乳腺特异性。这为在大动物中实施转基因提供了依据。     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomenain vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of lipid metabolism in murine.     

Heterologous CpG island becomes extensively methylated in the genome of transgenic mice

It is demonstrated that a heterologous (chicken) CpG island containing five Sp1 canonical recognition sequences becomes highly methylated in the genome of transgenic mice bearing one or several copies of the transgene. Similar levels of methylation of the chicken CpG island were observed in different tissues of transgenic mice except the brain where the level of methylation of this chicken CpG-rich fragment was significantly lower than in other tissues. Analysis of susceptibility of the "transgenic" CpG island to Hpa II and Msp I restriction nucleases revealed an unusual methylation pattern interfering with the action of both of these enzymes. A conclusion has been drawn that heterologous CpG island per se does not contain all necessary signals permitting to maintain its own non-methylated status in the genome of transgenic animals.     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomena in vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of     

人β_2m转基因小鼠的制备及鉴定(英文)

 制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性     

转hDAF基因小鼠的构建(英文)

本工作构建了含有ｈＤＡＦ基因的转基因小鼠,以便研究ｈＤＡＦ基因能否消除异种器官移植中的排斥反应。 采用ＤＮＡ重组的方法构建ｈＤＡＦ基因的表达载体ｐＳＰ６４ＨＰ（Ｆｉｇ．１）。通过受精卵显微注射,将其中的目的基因片段,转移到小鼠体内,建立转基因小鼠。再通过Ｄｏｔ ｂｌｏｔｔｉｎｇ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ杂交方法对出生小鼠的基因组特征进行查证。 连续两次对直接裂解菌液做ＰＣＲ扩增,筛选出重组质粒（Ｆｉｇ．２＆３）,酶切图谱（Ｆｉｇ．４）和Ｓｏｕｔｈｅｒｎ杂交（Ｆｉｇ．５）分析结果与预期吻合,出现预期条带;小鼠受精卵注射后存活比率为７７．９％,受精卵的发育率为３．４％,出生小鼠中,１０．５％出现清晰杂交信号。 表明：ｈＤＡＦ基因表达载体构建成功;并整合入小鼠基因组中。