粉尘螨Ⅰ类变应原瞬时表达载体的构建及其在烟草中的表达

目的 构建粉尘螨I类变应原瞬时表达载体TRBO-Der f 1并观察其在烟草中的表达。方法 以粉尘螨总RNA为模板,采用RT-PCR方法扩增Der f 1基因并定向克隆到瞬时表达载体TRBO中,转化根癌农杆菌（Agrobacterium tumefaciens）GV1301株后,抽提质粒进行酶切及PCR鉴定。将含TRBO-Der f 1重组质粒的GV1301注射本氏烟叶片,SDS-PAGE电泳检测注射3、4、5、6 d后Der f 1的表达,并进行Western blot鉴定。结果 电泳及测序表明Der f 1基因克隆成功,大小为627 bp。经PCR及酶切结果证实重组质粒TRBO-Der f 1成功转入根癌农杆菌。SDS-PAGE电泳检测表明,该蛋白于第5和6 d在烟草叶片中高表达,并通过Western blot得到验证。结论 粉尘螨I类变应原Der f 1成功表达于烟草叶片,为进一步研究源于植物的粉尘螨疫苗奠定了基础。     

恶性疟原虫裂殖子表面蛋白1C末端基因导入烟草叶绿体的研究

目的 将恶性疟原虫裂殖子表面蛋白1 C末端msp1-42基因（3D7株）导入烟草叶绿体基因组中并进行同质化筛选,为利用叶绿体表达系统生产MSP1-42蛋白提供基础材料。 方法　 利用烟草偏爱密码子设计克隆恶性疟原虫（3D7株）msp1-42基因的引物,从含msp1-42基因的pBluntmsp质粒中扩增出msp1-42,构建烟草叶绿体表达载体LRrrmsp。通过基因枪转化法转化烟草叶片,在500 mg/L壮观霉素的选择压力下筛选抗性植株,采用PCR鉴定抗性植株的msp1-42基因及aadA基因,对鉴定阳性植株进行同质化筛选（叶片切碎、在含500 mg/L壮观霉素分化培养基上分化出新的植株）3轮以上,并采用多重PCR分析其同质化情况。 结果　 构建了恶性疟原虫msp1-42基因的叶绿体表达载体LRrrmsp。基因枪转化后获得6个转化子,转化频率为0.6个/枪。转化3～5 d后,小块叶片开始增大增厚,并逐渐由绿色变为黄绿色,7～10 d后黄化或白化,再经约30 d的筛选培养,叶片上出现绿色小芽。PCR检测抗性植株的msp1-42及aadA基因,分别扩增出约900 bp与500 bp的条带,与预期相符。多重PCR分析从经过3轮同质化筛选植株的叶绿体基因中扩增出与未转基因烟草对照大小一致的弱条带,表明经过3轮同质化筛选的转基因植株仍含有未插入外源基因的叶绿体基因组。 结论 获得含恶性疟原虫msp1-42的烟草叶绿体表达载体,并将恶性疟原虫msp1-42基因导入烟草叶绿体基因组中,获得尚未完全同质化的转基因烟草。     

HBV表面抗原基因植物表达载体的构建及其在胡萝卜细胞中的表达

目的构建HBV表面抗原(HBsAg)的植物表达载体并在胡萝卜细胞中表达。方法以限制酶切M13/HB获得940bp含PreS2的HBsAg基因片段,将其插入到植物表达载体pBPC55,新质粒命名为pBPC91。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒pBPC93共同经基因枪(PDS-1000/He)转化胡萝卜悬浮细胞。经含除草剂(Bio-laphos)的MS液体培养基筛选,获得除草剂抗性胡萝卜细胞。结果纯化质粒pBPC91经酶切及序列分析表明HBsAg基因正确插入到E35S启动子下游且无密码子移位。对除草剂抗生胡萝卜细胞的组织学检测证实GUS基因有效表达;该细胞内DNA提取物PCR有940bp扩增带,蛋白萃取物的ELISA检测证实分别有HBsAg及PreS2-Ag蛋白表达。结论构建质粒pBPC91的PreS2及HBsAg基因可在转化胡萝卜细胞内表达。提示以植物细胞生产医用疫苗具有可行性。     

胸腺五肽基因转化烟草及其表达的研究

目的研究在烟草中表达胸腺五肽（Thymopentin，TP5）。方法合成TP5基因，构建重组载体pB1121＋TP5，以烟草叶盘为外植体，通过根癌农秆菌介导转染烟草，取卡那霉素抗性植株用PCR方法筛选转染阳性植株及RT—PCR、质谱检测阳性植株叶片外植体中的TP5表达。结果抗性植株整合并表达TP5。结论成功构建了含TP5的植物表达载体pB1121＋TP5，并能在烟草中表达。     

细粒棘球绦虫转基因植物载体重组pBI-Eg95-EgA31质粒构建及鉴定

目的构建并鉴定细粒棘球绦虫转基因植物载体重组pBI-Eg95-EgA31质粒。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后抽提总RNA为模板,采用RT-PCR方法分别扩增Eg95和EgA31编码基因,然后采用基因拼接法(gene SOEing)扩增Eg95-EgA31融合基因;将该融合基因定向克隆到植物表达载体pBI121中构建pBI-Eg95-EgA31重组质粒;电穿孔转化根癌农杆菌(Agrobacterium tumefaciens,At)LBA4404株,抽提质粒进行酶切及PCR鉴定。结果电泳及测序证实1 016bpEg95-EgA31融合基因克隆成功。经酶切及PCR证实重组质粒pBI-Eg95-EgA31成功转入根癌农杆菌。结论成功构建了细粒棘球绦虫转基因植物载体重组pBI-Eg95-EgA31质粒,为进一步构建细粒棘球绦虫转基因植物疫苗奠定了基础。     

pcDNA3?HBsAg?p30?ROP2真核表达载体的构建与鉴定

目的 目的 构建pcDNA3?HBsAg?p30?ROP2多基因重组表达载体, 并对其进行初步鉴定。方法 方法 根据重组体pcDNA3? p30?ROP2酶切位点和乙型肝炎表面抗原 （HBsAg） 基因序列等因素设计合成引物, 扩增HBsAg目的基因片段, 再应用酶切、 连接等分子生物学技术将HBsAg目的基因克隆至pcDNA3?p30?ROP2表达载体中。应用聚合酶链反应 （PCR） 初筛, 再采用 酶切、 测序等技术对构建的重组表达载体pcDNA3?HBsAg?p30?ROP2进行鉴定。 结果 结果 PCR扩增出HBsAg基因片段, 构建 了pcDNA3?HBsAg?p30?ROP2多基因真核表达载体。PCR与酶切结果显示, 该基因片段大小均与理论值相符; 测序结果显 示该重组表达载体包含了p30?ROP2和HBsAg目的基因的完整序列。 结论 结论 成功构建了多基因重组表达载体pcDNA3? HBsAg?p30?ROP2, 为进一步研究多基因核酸疫苗奠定了基础。     

抗汉滩病毒鼠源单抗3G1单链抗体转化拟南芥的研究

目的运用植物转基因技术,探索构建表达抗汉滩病毒鼠源单抗3G1单链抗体基因的植物生物反应器的可能性及技术路线。方法将鼠源单抗3G1单链抗体基因连接在双元表达载体pBI121CaMV35s启动子下游,构建了能在植物中高效表达的载体pBI121-3G1ScFv。利用根瘤农杆菌介导的抽真空转基因技术,将其转入野生拟南芥中。结果经过酶切分析、PCR验证及抗性筛选,结果表明3G1ScFv基因被导入拟南芥组织细胞,获得9株T0代转基因植株。结论本研究为进一步培育3G1ScFv遗传表达植株奠定了基础。     

口蹄疫病毒亚洲Ⅰ型YNBS/58株P12A-3C转基因植物双元表达载体的构建

目的双元载体是目前转基因植物工程普遍应用的载体，本文将口蹄疫病毒P12A基因、3C基因串联，构建植物表达载体pBin-438P12A-3G。方法为便于实验操作，本试验采用先将基因片段P12A、3C构建到一个中间质粒pcDNA3．1（＋）的策略，构建成pcDNA3．1（＋）-P12A-3C。结果通过对构建好的pBin438-P12A-3C（Mini—Ti）重组质粒进行PCR鉴定和序列测定，证明中间表达载体构建成功。结论通过三亲杂交法将重组质粒载体pBin438-P12A-3C导入到农杆菌GV3101，通过抗性筛选、PCR鉴定和序列测定，证明构建成功，与农杆菌中的help-Ti质粒共同组成植物双元表达载体，为以后的植物转化奠定基础。     

HBsAg诱饵载体的构建、表达及人胰腺cDNA文库的扩增、纯化和转化

目的构建HBsAg诱饵真核表达载体,并在AH109酵母菌中进行表达,同时扩增、纯化、鉴定及转化人胰腺cDNA文库。方法通过PCR扩增HBsAg基因,克隆到pGEM-T载体,测序正确后酶切并连接至表达载体pGBKT7中,转化酵母菌AH109,在色氨酸缺陷型培养基(SD/-Trp/Kana)上筛选阳性菌落,提取重组蛋白,经SDS-PAGE电泳后进行Western blot分析。扩增、纯化人胰腺cDNA文库并进行酶切鉴定及生物信息学分析,醋酸锂法将其转入Y187酵母菌。结果成功构建酵母表达载体pGBKT7-HBsAg、SDS-PAGE和Western blot显示重组蛋白在酵母细胞中正确表达;成功构建人胰腺cDNA文库,其容量为4×109 CFU/L,插入片段大小为0.5～2.0kb,长度不均,重组率为100%。结论成功构建HBsAg真核载体并在AH109酵母菌中表达,同时获得高质量的胰腺cDNA文库并转化入Y187酵母菌,为通过酵母双杂交筛选与HBsAg相互作用蛋白基因奠定了基础。     

乙型肝炎病毒S基因重组逆转录病毒载体的构建及其在真核细胞中表达

目的 探索逆转录病毒载体在基因治疗中的应用。方法 用ＤＮＡ重组技术构建ＨＢＶ－Ｓ基因重组逆转录病毒载体,电穿孔转染ＰＡ３１７后,筛选高表达克隆,用假病毒颗粒感染ＨｅｐＧ２、Ｐ８１５和ＥＬ４细胞,分别用ＲＴ－ＰＣＲ及ＥＬＩＳＡ法检测目的基因表达。结果 ＨＢｓＡｇ在上述细胞中获得不同程度的表达,细胞上清液（４８ｈ）中ＨＢｓＡｇ含量（吸光度值）分别为０．９２、０．０９和０．４７。结论 逆转录病毒用载体,     

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...     

乙型肝炎病毒-S基因重组逆转录病毒载体诱导小鼠体液和细胞免疫反应

探索逆转录病毒载体在基因治疗乙型肝炎中的应用。方法用ＤＮＡ重组技术构建ＨＢＶ－Ｓ基因重组逆转录病毒载体,电穿孔转染ＰＡ３１７后,用假病毒颗粒感染ＨｅｐＧ２、Ｐ８１５和ＥＬ－４细胞,并进行基因免疫。结果ＨＢＶ－Ｓ基因在上述细胞获得高效表达,在免疫动物体内产生高滴度抗一ＨＢＳ及有效的ＣＴＬ反应。结论该载体肌肉注射后有效激发对ＨＢｓＡｄｅ异性的体液免疫及细胞免疫反应,在细胞内稳定表达,是一种高效的基因转移系统,适用于基因治疗试验。     

Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg

AIM: To investigate the expression of recombinant HBsAg(rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests.METHODS: The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice‘s serum were examined by ELISA every week.RESULTS: By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo,level of rHBsAg expressed in leaves was the highest with the yield up to 300ng/g fresh weight. And the rHBsAg expression level in fruits was about 10ng/g fresh weight.In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response.CONCLUSION: The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale.     

pEGFP?N1?HBsAg?ROP2多基因重组表达质粒的构建及表达鉴定

目的 构建pEGFP?N1?HBsAg?ROP2重组质粒,并转染HEK293T细胞进行表达鉴定,为弓形虫病核酸疫苗的研制奠定基础。 方法 根据HBsAg基因序列和pcDNA3?p30?ROP2重组质粒酶切位点设计引物,PCR扩增HBsAg基因,经酶切、连接、转化,利用HBsAg基因替换p30基因,构建pcDNA3?HBsAg?ROP2重组质粒;经HindⅢ和KpnⅠ双酶切,将HBsAg?ROP2片段与pEGFP?N1真核表达载体相连,构建pEGFP?N1?HBsAg?ROP2重组表达质粒,转染HEK293T细胞,观察其蛋白表达水平。结果 HBsAg片段PCR产物约700 bp,与理论值相符;构建pcDNA3?HBsAg?ROP2重组质粒,双酶切电泳后得到约5.4 kb和1.9 kb的两条带,与预期结果相符。pEGFP?N1?HBsAg?ROP2双酶切后产生约4.7 kb和1.9 kb的条带,经测序鉴定,与GenBank发表的序列同源性为99.84%。目的基因已成功转染入HEK293T细胞中,且正确表达,蛋白浓度为3.08 mg/ml。结论 成功构建pEGFP? N1?HBsAg?ROP2重组表达质粒,转染HEK293T细胞能正确表达。     

辅助性T细胞表位HBV表面抗原融合基因真核表达质粒的构建

为提高HBVDNA疫苗的免疫效率，将一个通用型辅助性T细胞表位基因引入HBV表面抗原基因的5‘末端，构建成真核表达质粒。PCR方法合成PADRE-HBsAg目的基因，产物与pMDl8T载体连接，经Hind Ⅲ，EcoR Ⅰ双酶切，再克隆入真核表达载体pcDNA3．1( )。酶切及测序鉴定，该真核表达载体构建成功，为进一步观察其特异性细胞和体液免疫反应奠定了基础。     

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of α determinant region

AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice.METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD,respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA)and immune dot blotting to evaluate its antigenicity in vitro.The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the α determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal antiHBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106±26.7 vs1 814.4±776.3, P＜0.01,t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs6/10, 4.56±3.52 mIU/mL vs27.60±17.3 mIU/mL, P= 0.02, t= 2.7).CONCLUSIONS: HBsAg with deleted second-loop of the α determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.