乙型肝炎病毒包膜M蛋白在巴斯德毕德毕赤酵母中的表达

转化了乙肝病毒ｐｒｅＳ２－２基因的重组巴斯德毕赤酵母菌株经甘油培养基充分增殖,然后转移到甲醇培养基中进行诱导表达,破碎细胞并提胞内蛋白,经ＥＬＩＳＡ和ＷｅｓｔｅｎＢｌｏｔ检测证明有４株ＧＳ１１５／ＨＩ＾＋ＭＵＴ＾＋表达了ＨＢＶ Ｍ蛋白。     

不同培养基对SELDI-TO FMS细菌蛋白指纹图谱的影响

目的检测不同培养基对细菌蛋白SELDI-TOF MS指纹图谱的影响。方法 3株参考菌株分别接种在不同培养基中,利用SELDI-TOF MS检测蛋白指纹图谱,分析其异同。结果在所有培养基中参考菌株ATCC 12600、ATCC 25922和ATCC 27853蛋白质谱图恒定表达的蛋白峰个数为11、11、9个,同时菌株在不同培养基中也表达少量其独特的蛋白峰。结论比较不同培养基上生长的同株菌蛋白指纹图谱,显示在不同培养基其细菌特征蛋白恒定表达。     

A型产气荚膜梭菌α毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从A型产气荚膜梭菌标准株染色体DNA中扩增出α毒素基因,构建了含α毒素基因的重组菌株BL21(DE3)(pXETA02)。经酶切鉴定和序列测定证实,构建的表达质粒pXETA02含有α毒素基因序列。经SDS-PAGE、Western blot分析和ELISA检测,重组菌株表达的α毒素蛋白能够被α毒素单抗识别。表达优化结果表明,以IPTG为诱导剂诱导α毒素表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时α毒素蛋白表达量为34.83%。以乳糖为诱导剂诱导α毒素表达的优化条件是:培养基pH7.5、培养温度37℃,乳糖浓度0.1g/L,菌体生长密度OD600达到0.8时加入乳糖,诱导时间5h,α毒素蛋白表达量为23.82%。动物实验结果表明,用重组菌株α毒素蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。     

人67kD层粘连蛋白受体在毕赤酵母中的表达及活性分析

为实现人67kD层粘连蛋白受体(Human 67kD Laminin Receptor,67LR)蛋白的分泌表达,采用DNA重组技术将67LR cDNA片段插入分泌型酵母表达载体pPIC9K中,构建了相应的重组表达质粒pPIC9K-67LR并在GS115毕赤酵母菌株中表达,每升培养基经亲和层析可纯化目的蛋白12.56mg。纯化的目标蛋白能够与肺癌A549细胞竞争性结合其配体分子LN-1,具有相应的生物学活性,从而为深入研究人67LR的结构与功能奠定了基础。     

玉米赤霉烯酮降解酶毕赤酵母表达载体的构建及其表达

目的构建毕赤酵母表达载体pPIC9-ZEN-jjm,筛选高效分泌表达活性目的蛋白的菌株。方法克隆ZEN-jjm基因,经EcoRⅠ和NotⅠ双酶切连接至pPIC9中,电转化至毕赤酵母GS115。利用RDB培养基和甲醇诱导表达进行筛选。HPLC检测表达蛋白降解玉米赤霉烯酮的活性。结果测序表明ZEN-jjm成功插入pPIC9中,SDS-PAGE表明获得1株高效表达目的蛋白的重组酵母,其分子量约29 kDa。HPLC表明其能有效地降解玉米赤霉烯酮。结论玉米赤霉烯酮降解酶在毕赤酵母中获得了高效分泌表达。     

hPdx1和△-hPdx1蛋白表达系统的构建及功能鉴定

经鉴定成功构建了hPdx1和△-hPdx1蛋白表达系统,IPTG诱导蛋白表达后Western blot检测在37.2-52.3 kD之间出现了与两种目的蛋白理论分子量相符的蛋白条带。诱导后破碎菌体上清液,经分离纯化最终得到的目的蛋白纯度可达到95%以上。纯化的蛋白加入人胚胎干细胞培养基中1 h后通过细胞免疫荧光检测发现,hPdx1蛋白可进入细胞而△-hPdx1不能进入细胞,表明表达得到的蛋白具有生物活性。     

恶性疟原虫膜蛋白Pfs25在毕赤酵母中的组成型表达

目的:Pfs25蛋白是传播阻断型恶性疟疾疫苗的侯选抗原,在毕赤酵母中表达Pfs25蛋白,并对表达产物进行鉴定。方法:参照GenBank中公布的pfs25基因序列,通过毕赤酵母喜好密码子分析人工合成目的基因;采取定向克隆策略构建重组表达质粒pfs25/pGAPZαA,经BstXⅠ线性化,电转染法转化酵母菌株GS115,在Zeocin抗性的筛选培养基上获得表达目的基因的pfs25/pGAPZαA/GS115重组酵母菌,SDS-PAGE和Western印迹检测表达产物;通过在YPD培养基上传代培养和目的基因表达,验证重组菌株的遗传稳定性。结果:在毕赤酵母中表达了Pfs25蛋白,且重组菌株遗传性质稳定。结论:为研制基于Pfs25蛋白的传播阻断型恶性疟疾疫苗奠定了基础。     

毕赤酵母菌高效表达乙肝表面抗原的研究

目的:筛选高效表达HBsAg的毕赤酵母茵,制备目的蛋白.方法:从已确诊的乙肝病人血清中提取DNA,PCR扩增HBVS基因,将其分别克隆入毕赤酵母胞内表达栽体pPICZA中.构建重组质粒pPICZA-S和pPICZA-SH,经Sac I线性化后,LiCI化学法转化入酵母茵株GS115、X-33、KM71H和SMD1168.结果:诱导表达后的GS115工程茼单位体积的培养基所得的抗原含量最高,诱导培养基中加入0.1%酪蛋氨基酸后,可抑制目的蛋白的水解,有利于目的蛋白的表达,粗略估算表达量为15.3mg/L,最佳收获时间为72 h.结论:经SDS-PAGE和Westcrn-blot分析表明,所得产物为乙肝表面抗原S蛋白.     

优化的复合自动诱导培养基（CAI-4）对外源蛋白在大肠杆菌中 表达的影响

[目的]原核表达系统是目前最为广泛使用的一种外源蛋白表达系统。在利用原核表达系统表达目的蛋白的过程中,可溶性外源蛋白的产量是决定成本和效率的决定性因素。[方法]本项研究中利用本实验室构建的7种不同的重组质粒(-1、p-2、p-3、p-4、p-5、p-6、p-7),检测其在复合自动诱导培养基中的表达情况,评价哪一种培养基更适合于外源蛋白的表达,提高目的蛋白的产量。[结果]结果显示,这7种重组蛋白在复合自动诱导培养基的表达量是普通LB培养基的4~8倍;并在此基础上,对复合培养基的成份进行进一步优化,形成了一种优化培养基(改良培养基-4),P-1、P-2、P-3这3种融合蛋白在这种改良培养基中的表达量比优化前提高了至少2倍。     

通过共表达转铁蛋白和细胞周期蛋白D1改造CHO细胞株

目的:对现有的CHO-DG44细胞株进行改造,得到在无血清培养基中更具生长优势的CHO宿主细胞株。方法:分别克隆转铁蛋白和细胞周期蛋白D1基因,构建共表达2种基因的pIRES质粒,转染CHO-DG44细胞株,筛选G418抗性克隆。结果与结论:得到3株G418阳性克隆株,其中转铁蛋白和细胞周期蛋白D1表达水平最高的S6与CHO-DG44相比,在无血清培养基中生长更快、密度更高。     

TIMM29 interacts with hepatitis B virus preS1 to modulate the HBV life cycle

Hepatitis B virus (HBV), a major global health problem, can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinomas in chronically infected patients. However, before HBV infection can be adequately controlled, many mysteries about the HBV life cycle must be solved. In this study, TIMM29, an inner mitochondrial membrane protein, was identified as an interaction partner of the preS1 region of the HBV large S protein. The interaction was verified by both an immunoprecipitation with preS1 peptides and a GST-pulldown assay. Immunofluorescence studies also showed colocalization of preS1 and TIMM29. Moreover, it was determined that the preS1 bound with amino acids 92–189 of the TIMM29 protein. Infection of HBV in TIMM29-overexpressing NTCP/G2 cells resulted in a significant decrease of HBeAg and both extracellular particle-associated and core particle-associated HBV DNA without affecting cccDNA formation. Comparable results were obtained with TIMM29-overexpressing HB611 cells, which constitutively produce HBV. In contrast, knockout of TIMM29 in NTCP/G2 cells led to a higher production of HBV including HBeAg expression, as did knockout of TIMM29 in HB611. Collectively, these results suggested that TIMM29 interacts with the preS1 region of the HBV large S protein and modulates HBV amplification.     

HBV preS/S基因克隆及其在酿酒酵母中的表达

目的:探索利用酿酒酵母系统表达乙型肝炎病毒(HBV)preS／S基因。方法:利用PCR技 术,以HBV病毒DNA为模板,体外扩增HBV preS／S基因。然后构建重组表达载体pESC-preS／S。 用LiAc法转化酿酒酵母YPH50,选取重组菌进行培养,并诱导表达外源蛋白。提取蛋白浓缩后 进行SDS-PAGE分析,并经Western blot分析鉴定。结果:实验结果表明重组菌能够表达HBV preS／S蛋白。结论:利用酿酒酵母系统可成功表达HBV preS／S基因,为制备新型预防性疫苗提供 条件。     

Expression of the hepatitis B surface S and preS2 antigens in tubers of <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation. The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5 non-translated leader sequence, or the tuber-specific patatin promoter. HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters. The levels of preS2 antigen produced are the highest reported to date. Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

乙型肝炎病毒3′末端缺失的preS/S基因在转基因小鼠中的表达

从乙型肝炎病毒adr亚型的基因组DNA中分离3′末端缺失的preS/S基因的DNA片段,构建了由CMV启动子控制的真核表达载体。采用受精卵显微注射方法,获得了基因组整合有3′末端缺失的preS/S基因的2个转基因小鼠品系。在不同的时间点采取血清进行了ELISA分析,发现在这2个小鼠品系中3′末端缺失的preS/S基因可被表达,而且呈稳定状态。此小鼠品系的建立,对于探讨乙型肝炎病毒3′末端缺失的preS/S基因的表达产物在体内的生物学作用及其与肝细胞内细胞癌基因的转录激活之间的关系有重要意义。     

乙型肝炎病毒3’末端缺失的preS/S基因在转基因小鼠中的表达

从乙型肝炎病毒 ａｄｒ亚型的基因组 ＤＮＡ中分离 ３’末端缺失的ｐｒｅＳ／Ｓ基因的 ＤＮＡ片段,构建了由ＣＭＶ启动子控制的真核表达载体。采用受精卵显微注射方法,获得了基因组整合有３’末端缺失的ｐｒｅＳ／Ｓ基因的２个转基因小鼠品系。在不同的时间点采取血清进行了ＥＬＩＳＡ分析,发现在这２个小鼠品系中３’末端缺失的ｐｒｅＳ／Ｓ基因可被表达,而且呈稳定状态。此小鼠品系的建立,对于探讨乙型肝炎病毒３’末端缺失的ｐｒｅＳ／Ｓ基因的表达产物在体内的生物学作用及其与肝细胞内细胞癌基因的转录激活之间的关系有重要意义。     

Fatal exacerbation of type B chronic hepatitis triggered by changes in relaxed circular viral DNA synthesis and virion secretion

Virological features of fulminant liver disease-causing hepatitis B virus (HBV) have not been fully elucidated. We studied longitudinally the viruses obtained before and after fulminant liver disease in a patient with chronic HBV infection showing fatal exacerbation. HBV strains were obtained before and after exacerbation (designated as FEP1 and FEP2). Their virological features were investigated by in vitro transfection. FEP1 and FEP2 possessed higher activity of overall HBV DNA synthesis than the wild-type. FEP1 lacked competence for relaxed circular (RC) HBV DNA synthesis and RC HBV DNA-containing virion secretion, but FEP2 maintained it. Chimeric analysis revealed that the preS/S gene, where FEP1 had a considerable number of mutations and deletions but FEP2 did not, was responsible for impaired RC HBV DNA synthesis and virion secretion. Furthermore, incompetence of FEP1 strain was transcomplemented by the preS/S protein of wild-type strain. In conclusion, the viral strain after exacerbation showed resurgent RC HBV DNA synthesis and virion secretion, which was caused by conversion of the preS/S gene from a hypermutated to hypomutated state. This may have been responsible for disease deterioration in the patient. This is a novel type of HBV genomic variation associated with the development of fulminant liver disease.     

An anti-viral peptide derived from the preS1 surface protein of hepatitis B virus

The preS1 surface protein of the hepatitis B virus (HBV) is a key factor involved in initial viral entry into hepatocytes. It has been long postulated that an anti-HBV effect should be achievable using peptide fragments of the preS1. Recent reports demonstrated that several preS1-derived lipo-peptides in genotype D HBV exhibit nano to picomolar inhibitory activity against HBV infection. In this study, an acylated analog of a preS1 fragment, a 21-residue lipo-peptide (named 7524 BVS7) with a sequence of palmitoyl-GMGTNLSVPNPLGFFPDHQLDC-NH2, from genotype C HBV was produced base upon a previous structural study and was shown potently inhibits HBV infection with an IC(50) of approximately 20 nM.     

Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.     

Bacterial expression,purification, and in vitro N-myristoylation of fusion hepatitis B virus preS1 with the native-type N-terminus

Very low-level expression of hepatitis B virus (HBV) preS1 with the native-type N-terminus hampered the biochemical and functional studies on its myristoylation. In the present study, the fusion HBV preS1 with the native-type N-terminus and a His6-Tag fused to C-terminus (HBV preS1-HT) was highly expressed in Escherichia coli. This was due to an introduced mutation of the rare codon GGA found in the HBV preS1 to the codon preferred by E. coli, GGU. The protein was rapidly purified from bacterial lysate by Ni-IDA affinity chromatography. The experimental assays using 3H-labeled substrate demonstrate that the purified HBV preS1-HT can be effectively N-myristoylated by recombinant human protein N-myristoyltransferase (NMT) in vitro.