Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

Cloning of p15INK4b related gene CCRG and expression analysis in the renal carcinoma

AIM and METHODS:To analysis the factor that involved in renal carcinogenesis, we used the bait gene AK001518 to screen GenBank. To understand the relationship between cell cycle related gene(CCRG) and p15, we did RT-PCR and Northern Blot experiments. Then we examined CCRG expression level in renal carcinogenesis. RESULTS:Gained a function unknown gene CCRG that was 67% a mino acid identical with the gene AK001518 that was regulated by p15. It was shown that the CCRG mRNA was dramatically decreased when p15 gene was over-expressed. CCRG expression level was much higher in tumor tissues and cells than normal tissues and cells. CONCLUSION:The novel gene CCRG expressed highly in the renal carcinoma, which might play a significant role in the renal carcinogenesis.     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Influences of chronic hypoxia on the gene expression of Kv1.3,Kv2.1 and Kv3.1 induced by acute hypoxia in PASMC of rats

AIM and METHODS: Total RNA was extracted from 6th rat subcultured pulmonary artery smooth muscle cells(PASMC) exposed to continual chronic hypoxia or normoxia and the effects of chronic hypoxia on the changes of Kv1.3,Kv2.1,Kv3.1 mRNA in cultured PASMC induced by acute hypoxia were studied by semiquantitative RT-PCR in vitro. RESULTS:①Kv1.3,Kv2.1,Kv3.1 genes were found to be expressed in PASMC of rats exposed either to hypoxia or normxia.②The expression of Kv2.1 and Kv3.1 in 6th subcultured of PASMC in normaxia group could be upregulated by exposure to acute hypoxia,the levels of Kv2.1 and Kv3.1 mRNA were significantly increased from 0.646±0.092, 0.782±0.104 to 1.059±0.134, 0.985±0.116,respectively (P<0.01,n=5). ③PASMC cultured continuously in chronic hypoxia for 6 subcultures and then exposed to normoxia for 12 h,thereafter the expression of Kv2.1 and Kv3.1 were downregulated by acute hypoxia for 6 hours.The level of Kv2.1 mRNA was significantly decreased from 1.008±0.117 to 0.649±0.097 (P<0.01,n=5). CONCLUSION:Kv2.1,Kv3.1 genes might be oxygen sensitive genes.Chronic hypoxia might change the response of these Kv genes of PASMC to acute hypoxia and down-regulate its expression,which might probably decrease the role of Kv in HPV.     

IL-12 induces T lymphocytes apoptosis and influences the expression and signal transduction of Fas/FasL

AIM: IL-12 acts upon Tlymphocytes and activates its receptor complexes of β1/β2,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of Tcells and expression and signal transduction of Fas/FasLduring Tcell apoptosis. METHODS: The apoptosis of Tcells was detected by Annexin Vstaining cytometry and the expression of Fas/FasLunder different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic Tcell line(TIB-152) and the human lymphoma Tcell line(HTB-176) and the normal human Tcells to undergo apoptosis. The FasLexpression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasLexpression induced by IL-12 was inhibited by PKCinhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce Tcells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of Tcells. FasLparticipates in the progression of Tcell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasLexpression may be related with PKCpathway.     

Functional Analysis of ABC Transporter Genes From Botrytis cinerea Identifies BcatrB as a Transporter of Eugenol

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA–BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.     

植物苯丙氨酸解氨酶基因的表达调控与研究展望

苯丙氨酸解氨酶(phenylalanineammonia-lyase,PAL,EC4.3.1.5)是催化苯丙烷代谢途径第一步反应的酶,也是这个途径的关键酶,对植物有非常重要的生理意义。根据有关文献综述了植物PAL的分布与定位、酶学性质,总结了生长发育、钝化因子与调节因子、末端产物等内部因素及光、温、机械损伤与生长调节剂等外部因素对PAL的调控作用,得出外部因子是在转录水平上对酶活性实施调控的结论,并运用酶学和分子生物学方面的知识阐述了其调控机理。还着重阐述了PAL酶在果树上的研究现状与进展,并对其今后的研究及在果树上的应用进行了展望。     

差异显示法研究不同锌源对组织基因表达的影响

选用Avine肉用仔鸡作为试验对象 ,饲喂硫酸锌和蛋氨酸锌 2种锌源。采用银染差异显示技术进行差异基因的初步筛选。结果成功回收扩增差异条带 2 6条 ;其中加锌组 (硫酸锌组和蛋氨酸锌组 ) 2 3条 ,3条为缺锌组所特有。在加锌组的 2 3条中 ,6条为蛋氨酸锌组所特有 ,1 3条为硫酸锌组所特有 ,其余 4条为 2组所共有。这些初步筛选得到的差异基因尚需进一步进行克隆、测序和杂交进行验证。     

君子兰转化酶基因片段(CMCW1)的克隆和序列分析

 分析植物酸性转化酶基因的保守区序列，设计一对PCR引物，以君子兰基因组DNA为模板，采用PCR方法扩增出长约500 bp的DNA片段，克隆入pGEM-TEasy载体，测序结果表明获得君子兰酸性转化酶基因家族的一个成员CMCW1，该基因片段长518 bp，不含内含子，编码172个氨基酸。其序列已在GenBank中登记(登记号为AY151269)。在GenBank中进行同源性检索的结果表明，该成员编码的氨基酸与其它植物细胞壁酸性转化酶编码的氨基酸同源性较高。     

真空渗入法转pinⅡ基因菜薹外源基因的遗传与表达

 本研究对利用真空渗入法获得的转bar基因及pinⅡ基因菜薹，进行了外源基因的遗传及表达情况分析。结果表明通过该方法获得的转基因植株，外源基因能够稳定遗传。bar基因在 代的分离大部分符合孟德尔遗传规律，但也有不符合的群体；pin II基因在 代中稳定表达，但各个株系之间的表达水平差异较大，因而各个株系的抗虫效果也具有显著差异，即使具有相同外源基因拷贝数的各个株系，其PIN I／蛋白表达量和抗虫效果也具有显著差异。