去唾液酸糖蛋白受体特异性单链抗体的优化表达及亲和常数的测定

目的:表达及纯化抗去唾液酸糖蛋白受体(ASG-PR)的单链抗体的可溶性,并测定其亲和常数。方法:用噬菌体C1克隆感染E.coliHB2151,挑取单个菌落接种于2×TY培养基中,于37℃震荡培养过夜。将培养物作1∶100稀释并转种后,用终浓度为0.25、0.5、1.0mmol/L的IPTG,分别在37℃、25℃和20℃下诱导表达过夜。取其培养上清,用饱和硫酸铵沉淀后,以120g/LSDS-PAGE分析。另外,将饱和硫酸铵沉淀物用30mLPBS重新溶解、透析除盐后,用Ni2 螯合柱进行纯化,再以120g/LSDS-PAGE鉴定纯化scFvC1的纯度。用非竞争酶免疫法测定scFv的亲和常数。结果:用0.5mmol/LIPTG在25℃诱导过夜,表达的scFvC1的量较多,其相对分子质量(Mr)约为28000,以可溶性的形式存在于培养基中。通过Ni2 亲和柱纯化后scFvC1的纯度在95%以上,产量约为0.8mg/L。scFv的亲和常数为(2.31±0.36)×10-7mol/L。结论:以筛选的C1噬菌体感染E.coliHB2151后可表达低亲和力的可溶性scFv,对肝癌的基因治疗具有潜在的应用价值。     

中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域的原核表达及复性

目的对中国旱獭去唾液酸糖蛋白受体(ASGPR)H1和H2亚基糖基识别域(CRD)的克隆、表达、纯化及复性。方法用RTPCR从中国旱獭肝组织中扩增ASGPRCRDH1和CRDH2cDNA,分别将其克隆到原核表达载体pRSETB上,在埃希菌BL21(DE3)pLysS内诱导表达含6个组氨酸标签的融合蛋白。融合蛋白经Ni2 螯合柱亲和纯化后,在体外行透析复性。结果ASGPRCRDH1和CRDH2经原核表达后得到分子量约为22ku和15ku的目的蛋白,以包涵体形式存在。经Ni2 螯合柱亲和纯化后获得纯度大于95%的融合蛋白。利用仅识别天然构像的单克隆抗体对复性后产物进行检测,证明复性成功。结论成功地表达了具有活性的ASGPRCRDH1和CRDH2的融合蛋白,在肝脏疾病的靶向治疗研究中具有潜在的应用价值。     

可与肝癌细胞系HepG2特异性结合的单链抗体的表达及其特性

目的:在原核细胞中表达经噬菌体抗体库筛选可与HepG2细胞特异性结合的单链抗体(scFv),并对其特性进行鉴定。方法:采用正常肝细胞系L02和肝癌细胞系HepG2,从人源噬菌体抗体库中筛选可与HepG2细胞特异结合的scFv。对scFv DNA测序后,以其阳性噬菌体感染大肠杆菌HB2151,在IPTG诱导下表达scFv并以金属离子螯和层析(IMAC)进行纯化,用100g/L SDS-PAGE、Western blot及ELISA分析纯化的scFv。将纯化的scFv与HepG2细胞共孵育后,观察其对细胞生长的影响。结果:经筛选获得的2个相对特异性的scFv,分别命名为SLH04及SLH10。经IPTG诱导获得可溶性scFv的表达。Western blot分析证明,表达产物的相对分子质量(Mr)均为36000左右,与理论预期值相符。ELISA检测表明,纯化的scFv能与HepG2细胞特异性结合,并抑制HepG2细胞的生长。结论:从人源噬菌体抗体库中,筛选出能与HepG2细胞特异性结合的scFv,并在大肠杆菌中表达,有望用于肝癌生物治疗的实验研究。     

与肝癌细胞系HepG2特异性结合单链抗体的筛选与序列分析

目的：从人源噬菌体抗体库中筛选与肝癌细胞系HepC2特异性结合的单链抗体，为寻找肝癌细胞表面特异性标志及靶向研究奠定基础。方法：以正常肝细胞系L02为阴性筛选细胞，以肝癌细胞系HepG2为阳性筛选靶细胞，从人源噬菌体抗体库(Griflfin．l Library)经三轮筛选后，阳性菌质粒PCR确定其中含有单链抗体基因的克隆，通过细胞ELISA及FCM筛选特异性的单链抗体噬菌体，通过ABL3130全自动荧光测序仪测序，并通过GeneBank比对进行同源性分析，IPTG诱导可溶性单链抗体表达，并检测其与肝癌细胞株结合的特异性。结果：获得了2个特异性较高的阳性噬菌体单个克隆。经DNA测序后，在Genebank中与人的免疫球蛋白库进行比对，并用IMGTF／V-Quest软件进行分析，确定为2个插入序列不同的单链抗体片段。经细胞ELISA证明其阳性克隆菌培养上清可与肝癌细胞株特异性结合。获得的序列成功登陆Genebank，序列号为AY686498-AY686499。结论：从人源噬菌体抗体库中筛选到与肝癌细胞系HepG2特异性结合的具有功能活性的单链抗体，为进一步寻找肝癌细胞表面特异性抗原及靶向研究奠定了基础。     

中国旱獭去唾液酸糖蛋白受体糖基结合域的原核表达与多克隆抗体的制备

目的：构建中国旱獭去唾液酸糖蛋白受体（ASGPR）H1和H2亚基糖基识别域（CRD）的原核表达质粒，体外表达纯化后制备多克隆抗体。方法：RT-PCR扩增出中国旱獭肝组织中ASGPR CRDH1和CRDH2 cDNA，将其克隆至原核表达载体pRSET-B中，在大肠杆菌BL21（DE3）pLysS内诱导表达。用纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体，并采用酶联免疫吸附试验、Western blot及免疫组织化学检测抗体的灵敏度和特异性。结果：成功构建了中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域原核表达质粒pRSET-B.CRDH1和pRSET-B.CRDH2，目的蛋白可以高效表达，用其免疫BALB/c小鼠获得了高效价的特异性多克隆抗体。结论：首次成功表达了中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域多肽，且纯度高，免疫原性强，用其免疫小鼠获得的多克隆抗体特异性好、效价高，为在HBV感染模型-中国旱獭体内进行肝脏疾病的靶向治疗奠定了实验基础。     

抗恶性疟HRP-11单链抗体的筛选和鉴定

目的　研究开发简便、快速和有效的疟疾诊断技术。方法　利用噬菌体抗体库技术 ,在构建抗恶性疟原虫红内期噬菌体抗体库的基础上 ,经 3轮“吸附 -洗脱 -扩增”的富集反应后 ,筛选抗HRPII阳性克隆株并进行可溶性诱导表达 ,最后用ELISA和Westernblot等进行鉴定。结果　筛选出 6株抗HRPII阳性克隆株 ,表达的单链抗体Mr 为 310 0 0左右 ,能与HRPII抗原起特异性结合反应。结论　本研究为研制疟疾快速诊断试剂盒奠定了基础     

抗转铁蛋白受体抗体的筛选和鉴定

目的:从全人源噬菌体抗体库中筛选抗转铁蛋白受体(TfR)的抗体。方法:从HeLa细胞中提取总RNA,分两段反转录TfR胞外区cDNA,经两轮PCR后,获得胞外区基因。测序正确后,将其插入表达载体pPROEXTMHTa中,并在E.coliBL21(DE3)中表达。目的蛋白通过NiNTA金属螯合层析柱进行纯化、复性。用复性的TfR包被免疫管,从库容为1013cfu/L的全合成人源噬菌体单链抗体库中,筛选抗TfR的抗体。结果:得到约1.9kb的TfR胞外区基因,该基因以包涵体的形式在大肠杆菌中表达;利用纯化、复性的TfR从抗体库中筛选到了8个可与人TfR特异性结合的单链抗体。结论:利用TfR在大肠杆菌表达的复性产物,可以筛选到与天然TfR特异性结合的单链抗体。     

抗人肺癌单链抗体噬菌体库的构建及特异性单链抗体的鉴定

目的 构建人源噬菌体抗体库,并从中筛选出抗肺癌人源单链抗体。方法 提取肺癌患者癌旁淋巴结组织,通过RT-PCR扩增出重链可变区基因（VH）和轻链可变区基因（VL）,再经剪切-重叠-延伸PCR(SOE-PCR)将VH 和VL连接得到单链抗体（ScFv）。将双酶切后的ScFv基因片段克隆入噬菌体表达载体pCANTAB5E,得到初级噬菌体抗体库。以肺腺癌细胞株A549为抗原对抗体库进行4轮筛选富集,鉴定抗体库性能。将得到的阳性克隆用IPTG诱导表达并进行检测。结果 成功构建噬菌体单链抗体库。经筛选富集,噬菌体收获率得到增加,第4轮是第1轮的115倍。随机选取10个克隆,通过ELISA法检测到其中7个与A549细胞呈阳性反应,阳性率为70%。SDS-PAGE及ELISA检测证实得到人源抗肺癌单链抗体。结论 成功构建人源单链抗体噬菌体库,从中获得具有较高特异性的抗人肺癌单链抗体。     

人源性bFGF单链抗体的筛选、表达及鉴定

目的 用重组人碱性成纤维细胞生长因子(rhbFGF)筛选大容量人源噬菌体抗体库,获得能与bFGF特异性结合的人源性单链抗体(scFv).方法 用rhbFGF对大容量人源噬菌体抗体库进行4轮"吸附-洗脱-扩增"筛选,并通过phage ELISA法对随机挑选的克隆进行抗原结合活性测定,得到的阳性克隆进一步进行原核可溶性表达及鉴定.结果 从104个随机挑选的克隆中,筛得39株抗bFGF的抗体,通过对抗体可变区基因进行多样性分析,发现存在8种不同抗体可变区基因,挑选其中8株phage ELISA显色最高的克隆进行酶切和测序鉴定,发现其中7株为正确的抗体基因序列,通过大肠杆菌成功的进行了scFv的可溶性表达,通过竞争ELISA发现其中一株scFv能够抑制bFGF与其高亲和力受体FGFR1βⅢC的结合.结论 通过筛选噬菌体抗体库获得到7株抗bFGF特异性抗体,其中一株抗体能够抑制bFGF与其高亲和力受体FGFR1的结合.     

抗TSLP全人源单链抗体筛选与初步鉴定

目的:表达TSLP蛋白,从全人源单链抗体文库中筛选得到抗TSLP单链抗体。方法:扩增TSLP cDNA,将目的基因与表达载体pET101/D-TOPO连接,转化大肠杆菌BL21,IPTG诱导表达并对表达产物进行纯化鉴定。以生物素化的TSLP蛋白为抗原从前期构建的全人源抗体文库中采用噬菌体展示技术筛选抗TSLP全人源单链抗体(scFv)。结果:扩增的TSLP cDNA片段大小为423 bp左右。表达的TSLP蛋白大小为26 kD左右,Dot blot及Western blot鉴定显示表达的蛋白正确,为TSLP目的蛋白。以生物素化的TSLP蛋白为抗原,采用噬菌体展示技术对全人源抗体文库进行3轮富集,通过ELISA检测有35%的scFv与TSLP有结合特性。将筛选的与TSLP结合能力强的单链抗体进行了Western blot鉴定和测序。结论:成功筛选到抗TSLP全人源单链抗体。     

Comprehensive sequence analysis of HLA-DQA1 and -DQB1 alleles and characterization of DRB1-QAP-DQA1-DQB1 haplotypes

It is well known that both chain and β chain of HLA-DQ are highly polymorphic. However the polymorphisms outside the hypervariable region were not fully examined so far. To further clarify the polymorphisms in DQ genes, we determined the nucleotide sequences of full length cDNA, spanning from the leader sequence to the stop codon, from 15 DQA1 alleles and 15 DQB1 alleles. We identified several new DQ alleles which had identical exon 2 sequence and were different in other exons. On the basis of the sequence analyses, a comprehensive PCR-based oligotyping system for DQA1 gene was established. We then characterized DRB1-QAP(DQA1 promoter)-DQA1-DQB1 haplotypes of B-lymphoblastoid cell lines homozygous for HLA and healthy unrelated Japanese and Norwegian populations. It was revealed that DQA1 alleles, which were identical in exon 2 but different in other exons, showed close linkage disequilibrium with diferent characteristic DRB1, QAP and DQB1 alleles. These results suggest that DR-DQ haplotypes have been generated in the early stage of molecular evolution.     

Hepatoprotective activity of 1-ethoxysilatrane and 1-isopropoxygermatrane

1-Ethoxysilatrane and 1-isopropoxygermatrane stimulated liver regeneration via activation of some components of the protein-synthesizing complex, in particular, aminoacyl-tRNA synthetases. Acylating activity of aminoacyl-tRNA synthetases increased. Possible mechanisms of these changes are discussed.     

AhR途径,CYP1A1、CYP1B1,雌激素代谢及作用过程中的调节

多环芳烃和多卤化烃是环境中广泛分布的有害物质,可通过与细胞芳烃受体结合,从而影响外来化合物代谢酶系如细胞色素氧化酶P450 1A1、1B1的表达,并通过这些酶的催化作用调控雌激素的代谢及作用,进而部分决定了雌激素对机体的作用效应。上述复杂的过程可受到多种因素的影响。     

Polymorphisms of CYP1A1 and GSTM1 genes and susceptibility to oral cancer

PURPOSE: Oral cancer is the fifth most common form of cancer in the world and comprises 6.5% of all cancer deaths. Since one of the major risk factors for oral cancer is tobacco use, we hypothesized that polymorphic genes coding for tobacco carcinogen-metabolizing enzymes may play a role in oral cancer susceptibility. MATERIALS AND METHODS: To investigate the association between polymorphisms of the CYP1A1 and GSTM1 genes and risks for oral squamous cell carcinoma (OSCC) in the Korean population, the prevalence of the CYP1A1 Mspl and GSTM1 null polymorphisms were examined in 72 patients with histologically confirmed primary OSCC, as well as in 221 healthy control subjects. RESULTS: A significant risk increase for oral cancer was observed among subjects with the homozygous CYP1A1 (m2/m2) genotype (OR=3.8, 95% CI=1.9-7.7), but not the GSTM1 null genotype (OR=0.7, 95% CI=0.4-1.3). Risk for oral cancer was significantly increased in subjects with the homozygous CYP1A1 (m2/m2)genotype, regardless of smoking history (smokers; OR=4.4; 95% CI=1.2-16.3; non- smokers OR=4.9; 95% CI=1.9-12.5). Using the potentially most protective genotype GSTM1 (+)/CYP1A1 [(m1/m1)+ (m1/m2)] as the reference group, an increased risk for oral cancer was observed among subjects with the GSTM1 (+)/ CYP1A1 (m2/m2) (OR= 2.0, 95% CI=0.8-5.2), and GSTM1 (-)/ CYP1A1 (m2/m2) (OR=4.9, 95% CI=1.5-15.5) genotypes (p < 0.009, (chi2 trend test). CONCLUSION: Our results suggest that individuals with a genotype of CYP1A1 (m2/m2) and GSTM1 (-) are highly susceptible for OSCC and that the CYP1A1 (m2/m2) genotype is closely associated with increased risk of OSCC in Koreans.     

Initial Changes of Rat Leukemia Induced by 1-Ethyl-1-Nitrosourea and 1-Butyl-1-Nitrosourea

The initial histological changes of leukemia were investigated in rats to which 1-ethyl- 1-nitrosourea and 1 butyl 1 nitrosourea were orally administered. The appearance of orthochromatic erythroblasts in the peripheral blood was used as the index of the initial stage of leukemia. The rat leukemia progressed from solitary lesions to scattered and further diffuse lesions. These leukemias are thought to begin as one, or only a few nodular foci, mainly in the bone marrow and partly in the spleen. Acta Pathol Jpn 42: 158–165, 1992.     

Maternal genetic polymorphisms in CYP1A1, GSTM1 and GSTT1 and the risk of hypospadias

Hypospadias is one of the most common congenital anomalies. Increased exposure to environmental factors (endocrine-disrupting chemicals and smoking) or maternal endogenous estrogen may cause hypospadias because male sexual differentiation is dependent on normal androgen homeostasis. Moreover, interactions between genetic factors and cigarette smoking and other chemicals have been suggested. It has been demonstrated that the CYP1A1 metabolizes not only environmental chemicals but also estrogens, and glutathione-S-transferases (GSTs) are detoxification enzymes that protect cells from toxicants by conjugation with glutathione. In this study, to investigate the association of CYP1A1 (MspI), GSTM1 and GSTT1 polymorphisms with hypospadias, a case-control study of 31 case mothers who had boys with hypospadias and 64 control mothers was performed in Japan. These polymorphisms were investigated by PCR-based methods using DNA from peripheral lymphocytes. We found that the heterozygous CYP1A1 and heterozygous and homozygous CYP1A1 were less frequent in the case mothers than in the control mothers [adjusted odds ratio (OR)=0.17, 95% confidence interval (CI)=0.04-0.74, OR = 0.28, 95% CI = 0.08-0.97, respectively]. We found no effect of maternal smoking on the hypospadias risks among the gene polymorphisms. The results suggest that mothers with the CYP1A1 MspI variant allele may have a decreased risk for hypospadias.     

Distribution of HLA-DRB1, -DQA1 and -DQB1 alleles and DQA1-DQB1 genotypes among Norwegian patients with insulin-dependent diabetes mellitus

We have studied 87 unrelated Caucasian insulin-dependent diabetes mellitus (IDDM) patients and 181 healthy controls by oligotyping for 20 DRB1, eight DQA1 and 13 DQB1 alleles, and established their DR-DQ haplotypes and DQ genotypes. An increase of DRB1 alleles encoding DR4 was found among IDDM patients, but the distribution of DR4 subtypes did not differ among DR4-positive IDDM patients and controls. The frequency of certain DRB1-DQA1-DQB1 haplotypes and DQA1-DQB1 genotypes was significantly increased among IDDM patients. Taken together, the data suggest that IDDM is primarily associated with several (at least five) different DQ alpha beta heterodimers.     

先兆子痫患者HLA-DQA1、-DQB、-DPA1基因多态性

目的：探讨人类白细胞抗原HLA-DQA1、-DQB1、-DPA1基因多态性与先兆子痫发病的关系。方法：采用序列特异性引物技术（PCR-SSP） 对46例先兆子痫患者和105例正常孕妇及其新生儿进行HLA-DQ-DPA1等位基因分型。结果：所有标本共检出11种HLA-DQA1基因表型、16种HLA-DQB1基因表型、6种HLA-DPA1基因表型。先兆子痫患者HLA-DQB1＊0301基因频率高于正常孕妇，差异有显著性（Pc=0．032，RR=2．43，AR=0．30），其余各基因表型频率两组比较差异均无显著性。结论：HLA-DQB1＊0301基因可能是一种先兆子痫发病的易感基因。     

Gene polymorphism of HLA-DPB1 and DPA1 loci in caucasoid population: Frequencies and DPB1-DPA1 associations

The genetic polymorphism of the HLA-DPB1 and DPA1 loci was studied in 60 unrelated caucasoid individuals by PCR-RFLP. The polymorphic second exon of DPB1, the third exon of DPA1, and the trans-membrane DPA1 exon were specifically amplified in vitro by polymerase chain reaction (PCR). Amplified DNAs were digested with selected enzymes. Twenty patterns were obtained with DPB1 defining 20 DPB1 alleles. Thirty-nine homozygous cell lines were used as HLA-DP reference cells. The results obtained with these cell lines were compared to those obtained by PLT, RFLP, and SSO. Although three subdivisions of the allele DPA1^*01 were reported, DPA1^*0103 was the only represented one in the caucasoid population. In the studied population, it was the most frequent DPA1 allele (76.6%), whereas DPA1^*0201 frequency is 23.3%. DPB1^*0401 and DPB1^*0402 are the most frequent among the DPB1 alleles (40.0% and 13.3%, respectively). This may lead to a lower HLA-DPB1 diversity among caucasoids. Certain HLA-DPB1 alleles associate exclusively with one DPA1 allele (DPB1^*0401, 0402, and 0301 with DPA1^*01 and DPB1^*0101, 0501, and 1701 with DPA1^*0201) whereas the others can associate with both DPA1 alleles. This by itself can create another kind of polymorphism, indicating the importance of HLA-DPA1 typing. Thus, PCR-RFLP seems to be one of the best DNA typing methods: it represents direct, accurate, fast, and nonradioactive typing for both HLA-DPA1 and HLA-DPB1 alleles.