Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.     

Expression of biologically active human clotting factor Ⅸ(hFⅨ) in the mammary gland of transgenic mice

The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.     

Specific anti-tumor effect induced by attenuated <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1＋ VEGFR2（n1-7） was transformed into competent attenuated Salmonella typhimuriurn SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1＋VEGFR2（n1-7）. To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy （TEM） was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an enhanced accumulation of CD8^＋ cytotoxic T lymphocytes, as well as an increase in CD4^＋ cells in the tumore of animals treated with the oral gene vaccine compared to tumors from control group mice. UI- trestructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the r     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.     

Regulatory effect of cotton leaf curl virus AC2 on virion sense promoter

The AC2 gene of cotton leaf curl virus (CLCuV) was obtained by polymerase chain reaction (PCR) . The total DNA of the CLCuV infected tomato leaves was used as template, and the amplified DNA fragment was inserted into a cloning vector. Transient expression vectors were constructed by inserting the AC2 gene into downstream region of CaMV 35S promoter. These constructs were delivered into tobacco and cotton leaf cells for transient expression by particle bombardment. The results indicated that the virion sense promoter was activated by AC2 and its activity increased remarkably. However, the activity of transactivated virion sense promoter was still lower than that of the complementary sense promoter. The expression pattern of transactivated virion sense promoter was similar to that of the complementary sense promoter, namely with high activity in both mesophyll and vascular tissues. The possibility of application of AC2 in plant genetic manipulation was also explored.     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest titers up to 8.5×10⁸ cfu·mL^-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P＜0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P＜0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5±6.1) ng·mL^-1. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was not detected by the method of Bethesda, no germ line transmission and any side effects associated with gene transfer were found. Our results indicated that neonatal gene therapy for hemophilia B mice by VSV-G pseudotyped retrovirus is promising.     

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

Objective： To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein （Ad-GFP） into the primary cultures of fetal neural stem cells （NSCs） by the expression of GFP. Methods： The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results： After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion： We have success- fully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.     

小鼠造血干细胞的分离、培养及重组慢病毒载体介导的离体hFⅨ基因表达

造血干细胞是基因治疗理想的靶细胞之一,尤其适用于遗传性血液病.而重组慢病毒载体能高效感染造血干细胞,成为造血干细胞途径基因治疗的理想载体.从小鼠骨髓细胞中分离出单个核细胞(MNCs)进行体外悬浮培养,并用免疫磁珠法分离得到高纯度的小鼠Lin-CD117+造血干细胞(HSCs).体外悬浮培养期间,添加细胞因子的造血干细胞的细胞数和集落数逐渐增加,而未添加细胞因子的对照组的细胞数量无明显增加,细胞集落递减.用磷酸钙介导的共转染法制备了携带FⅨ基因的FUXW重组慢病毒,用慢病毒载体分别感染从ICR小鼠和C57小鼠中分离得到的MNCs,7d后测得细胞上清中hFⅨ的表达量分别为41.7±4.2和34.5±6.6ng/mL,而慢病毒感染C57小鼠造血干细胞,添加细胞因子组上清中hFⅨ的表达量为46.6±5.7ng/mL,不添加细胞因子组为33.3±4.8ng/mL.实验结果表明,重组FUXW慢病毒载体可有效感染小鼠单个核细胞和Lin-CD117+造血干细胞,添加细胞因子可提高转移基因的表达量.     

Npas4基因过表达慢病毒的构建及其在SK-N-SH细胞的表达

研究旨在构建Npas4基因过表达慢病毒,为进一步深入探索Npas4基因的功能奠定基础。用人工合成大鼠Npas4基因c DNA片段,将其插入p CDH-CMV-MCS-EF1-cop GFP构建慢病毒表达质粒p CDH-Npas4。酶切、测序验证质粒后,将p CDHNpas4和辅助质粒共转染包装细胞293T,浓缩上清得病毒颗粒并测定病毒滴度。取病毒颗粒感染SK-N-SH细胞48 h,收集细胞采用Western blotting法检测Npas4蛋白的表达。p CDH-Npas4携载正确Npas4基因,将其包装293T细胞后能产生病毒。病毒滴度为1.05×109TU/m L。相比于转染GFP病毒对照组(GFP)和未转染对照组(control),Npas4重组慢病毒组(Npas4)的细胞Npas4蛋白表达显著增高。成功构建Npas4基因过表达的重组慢病毒载体p CDH-Npas4,并获得高效的重组慢病毒,能将外源Npas4基因导入SK-N-SH细胞,为进一步研究Npas4基因的相关功能奠定了基础。     

Preparation of rAAV/hFⅨ by HSV/AAV hybrid helper virusand evaluation of its safety

Hemophilia B is a hemorrhagic disease resulting from Factor Ⅸ gene (hFⅨ) mutation as an X-linked recessive inherited trait. The incidence of this disease is 1 in 30000. Clinical treatments depend mainly upon blood transfusions or administration of prothrombin complex so that patients are at the risk of infections with the HIV and hepatitis viruses. Gene therapy offers an attractive alternative in the treatment of hemophilia B by eliminating those risks. In 1991, our lab conducted clinica…     

汉滩病毒囊膜糖蛋白g2基因重组腺病毒的构建与表达

获得汉滩病毒G2 基因 ,构建其重组腺病毒并在HEK2 93细胞中包装表达 ,为研究汉滩病毒基因疫苗提供了实验基础。设计引物采用PCR从含汉滩病毒 \|76 1 1 8株M基因的M5 6质粒扩增出糖蛋白G2 基因片段 ,并将其克隆入腺病毒载体Adeno XviralDNA ,筛选获得重组腺病毒DNA ,转染HEK2 93细胞 ,包装、扩增后得到汉滩病毒G2 基因重组腺病毒原种 ;并在感染细胞内初步表达 ,用ELISA检测表达产物。得到了含汉滩病毒G2 基因的重组腺病毒 ,其滴度约为 1 0 10 pfu/mL ,同时在感染的HEK2 93细胞中检测到汉滩病毒糖蛋白G2 的表达。含汉滩病毒糖蛋白G2 基因重组腺病毒的成功构建 ,为研究汉滩病毒基因疫苗提供了实验基础     

BGC-823细胞中慢病毒途径针对HIF-1α基因的RNAi实验

目的：构建人HIF-1α基因的shRNA慢病毒载体、包装生产重组慢病毒颗粒,病毒途径高效感染胃癌细胞株BGC-823,并验证基因沉默效率。方法：在NCBI数据查找人HIF-1α基因序列,然后使用siRNA在线设计软件,设计针对基因CDS区的3条siRNA序列及Negative序列。根据设计好的siRNA序列设计双链互补的shRNA-Oligo DNA,退火形成双链后与线性化载体链接,构建shRNA重组表达载体。经由293TN细胞包装shRNA重组慢病毒颗粒,随后使用重组病毒感染BGC-823,通过荧光标记蛋白GFP确定感染效率后收集细胞样本,分别采用Real-time PCR和Western blot方法检测靶基因在mRNA和蛋白质水平的沉默效率。结果：shRNA重组表达载体测序结果与设计序列完全一致,包装病毒后滴度达到1×104 ifu/μL。慢病毒感染BGC-823细胞取得了极高的基因转导效率。mRNA检测结果显示,siRNA3对于对与目的基因的沉默效果最好,较阴性对照序列组相比较,mRNA的表达量下降了92%,Western blot检测的结果与mRNA检测结果完全相符合。结论：通过慢病毒途径,可以在BGC-823细胞中高效的进行HIF-1α基因的沉默。     

慢病毒介导的HPV16-E7基因RNAi细胞模型的建立

 建立HPV16-E7基因的RNAi细胞模型,可为进一步研究HPV16-E7蛋白作用及致癌机制提供物质前提。选择HPV16-E7基因特异的siRNA片段,构建慢病毒siRNA重组表达载体,经293FT病毒包装细胞转染产生重组病毒,并以此感染HPV16阳性SiHa宫颈癌细胞、抗菌素筛选和分子生物学鉴定,建立了稳定表达HPV16-E7-siRNA的RNAi细胞模型。该细胞模型,对目标基因（E7）表达的抑制效率,也以蛋白免疫印迹和RT-PCR确证。由此得出,利用慢病毒载体和HPV16-E7基因特异的siRNA片段,可以建立一种稳定、有效和可行的RNAi细胞模型,为进一步研究E7蛋白的作用及致癌机制奠定了重要的物质基础。     

梅花鹿鹿茸软骨细胞和间充质干细胞永生化细胞株的构建及鉴定

分离梅花鹿鹿茸软骨细胞和间充质干细胞,使用SV40 LT抗原慢病毒载体建立永生化软骨细胞和间充质干细胞系并鉴定。将含有SV40 LT基因片段的慢病毒载体,转染人胚肾细胞293T获得包装后的病毒粒子,感染鹿茸软骨细胞及间充质干细胞,连续传代培养,通过形态观察、细胞增殖、real-time PCR、甲苯胺蓝染色法和流式细胞术等方法检测SV40 LT抗原表达以及细胞性质。实验所建立的永生化鹿茸软骨细胞系与间充质干细胞系能够稳定传代并具有较强的体外增值活性。RT-PCR检测到SV40T抗原的表达,通过甲苯胺蓝染色法和流式细胞术鉴定所得细胞系具有原代细胞的基本性质。成功获得永生化的软骨细胞与间充质干细胞系,为后续鹿茸生长及发育研究奠定了基础。     

表达大鼠FoxA1的慢病毒制备和鉴定

通过PCR扩增获得大鼠FoxA1的cDNA,构建慢病毒重组载体plv-rFoxA1-IRES-EGFP,并瞬时转染293T细胞观察绿色荧光蛋白(GFP)的表达及Western blot检测FoxA1的表达.通过钙转将该重组载体与辅助质粒△8.91,pVSV-G共转染293T细胞制备慢病毒.研究结果表明,大鼠FoxA1慢病毒质粒成功构建,并且证实所制备的慢病毒能感染细胞,使细胞有效表达FoxA1蛋白,为FoxA1的功能研究奠定了材料基础.     

鲤IGF2b基因内含子的克隆、基因组序列分析及慢病毒载体构建

 为了了解鲤IGF2b基因与鲤生长性状之间的关系,以建鲤为试验材料,克隆了IGF2b基因内含子,分析其基因组序列的特点,构建了IGF2基因的慢病毒载体,同时观察其在293T细胞中的表达活性。结果获得鲤IGF2b 5 173 bp长度的基因组DNA序列(HM755899),共有3个内含子,4个外显子;在3′非翻译区存在2个CpG岛,在5′端非翻译区存在(T)n重复序列;除此之外,成功构建了慢病毒载体质粒Lenti-IGF2b-IRES-EGFP,转染293T细胞后,产生的重组慢病毒颗粒出现高表达绿色荧光,荧光定量PCR检测发现IGF2b基因在293T细胞中高表达。这些结果将为研究IGF2b基因在多态性、表达方面与鲤生长性状之间的关联奠定基础。