Another example of a KEL1 variant red cell phenotype due to a threonine to serine change at position 193 of Kell glycoprotein

BACKGROUND: Healthy subjects whose red blood cells (RBCs) react variably with anti-KEL1, but strongly express other Kell blood group antigens, have been described and called KEL1 variant. A 53-year-old Caucasian blood donor was identified whose RBCs reacted with three monoclonal and two polyclonal anti-KEL1 and did not react with two monoclonal and one polyclonal anti-KEL1. The molecular basis of this phenotype was investigated.
STUDY DESIGN AND METHODS: Genomic white blood cell DNA was analyzed for KEL * 1/2 genotype by utilizing sequence-specific primers and polymerase chain reaction. In addition, the region of the KEL * 1/2 polymorphism at position 578 of KEL was analyzed by DNA sequencing.
RESULTS: Genotyping of the donor with the KEL1 variant phenotype revealed that he was KEL * 2 homozygous. Sequencing revealed one typical KEL * 2 allele and a KEL * 2 allele with an adenosine (A) to thymidine (T) substitution at position 577 that predicted a threonine to serine change at position 193.
CONCLUSION: It is not known if this phenotype is clinically relevant, but for at least some genotyping applications probes that identify this polymorphism should be used and anti-KEL1 should be tested for reactivity to this allele.     

Genotyping of KEL1 and KEL2 of the human Kell blood group system by the polymerase chain reaction with sequence-specific primers

BACKGROUND: Kell is a major antigenic system in human red cells, with more than 20 identified antigens. KEL1 and KEL2 are two opposing low- and high-frequency alleles. Immunization to KEL1 is clinically significant, because anti-KEL1 can cause severe reactions to transfusion of incompatible blood, as well as hemolytic disease of the newborn. At the nucleotide level, the difference between the KEL2 and KEL1 alleles is a single-base change within exon 6 that results in the substitution of methionine (ATG) for threonine (ACG) at position 193. STUDY DESIGN AND METHODS: An assay using polymerase chain reaction and sequence-specific primers to genotype for the KEL1 and KEL2 alleles has been developed. It uses two allele-specific forward primers for either KEL1 or KEL2 and a single reverse-consensus primer. RESULTS: A validation study of 42 serologically typed samples (5 KEL:1,-2 [K+k-]; 23 KEL:1,2 [K+k+]; and 14 KEL:-1,2 [K-k+]) was performed. A concordance rate of 100 percent (42/42 samples) was observed between polymerase chain reaction with sequence-specific primers and serologic typing. CONCLUSION: This rapid, nonradioactive, Kell system genotyping assay does not require the additional steps of probe hybridization or restriction enzyme digestion. This application of polymerase chain reaction with sequence-specific primers should prove particularly useful in Kell system genotyping of amniotic cells to identify pregnancies at risk for hemolytic disease of the newborn.     

Transfer and expression of the gene encoding a human myeloid membrane antigen (gp150).

DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.     

CDCP1胞外段基因的克隆及在大肠杆菌中表达

目的：构建携带 CDCP1胞外段基因的原核表达载体。方法以 A549的 mRNA 为模板,应用所设计的引物通过 RT-PCR 法扩增 CDCP1胞外段基因;将 PCR 产物与 pGEX-KG 载体连接,获得重组质粒 pGEX-KG/CDCP1;经双酶切、PCR 及 DNA 序列测定进行鉴定;IPTG 诱导表达。结果①RT-PCR 扩增得到约270 bp 大小的 CDCP1胞外段基因目的片段;②目的片段正确插入到 pGEX-KG 中;③经 IPTG 诱导表达,可见在相对分子质量约35kD 处出现明显的诱导蛋白条带,与预期一致。结论成功构建了携带 CDCP1胞外段基因的原核表达载体,在大肠杆菌中获得大量表达。     

K23. A low-incidence antigen in the Kell blood group system identified by biochemical characterization

An antibody in the serum of a gravida 4, para 3 woman reacted with red cells from two of her children, her husband, and his mother, but with none of more than 2100 reference red cell samples and blood samples from donors. The reactive antigen was inactivated by 2-aminoethylisothiouronium bromide or dithiothreitol-papain treatment. The antigen was immunoprecipitated from paternal red cells with maternal antibody and shown to migrate by sodium dodecylsulfate polyacrylamide gel electropheresis as a single protein of approximately 93,000 daltons. After transfer to nitrocellulose paper by Western blotting, the protein reacted with a rabbit antibody specific for Kell protein. The chemical inactivation and electrophoretic findings were characteristic of Kell group antigens. The reaction with the rabbit antibody establishes that the "new" low incidence antigen was an epitope on Kell group protein and must be coded for by the Kell gene. It has been designated K23.     

Variations in the expression of granulocyte antigen NB1

BACKGROUND: Between 87 and 97 percent of whites express NB1 alloantigen on some but not all of their granulocytes. The expression of NB1 has not been compared among large groups of adults of different sexes, ages, and ethnic groups. Previous testing of whites suggests that the expression of NB1 is variable. STUDY DESIGN AND METHODS: Serologic testing of granulocytes from 224 persons with two examples of MoAb to NB1 (1B5 and 7D8) was performed to distinguish phenotypic differences among age, sex, and ethnic groups and differences in reactivity to MoAbs. The donors were from 17 to 82 years of age, and 87 were female. They were from four ethnic backgrounds: 54 were African American (black), 10 were Asian, 9 Hispanic, and 152 white. Granulocytes were tested by flow cytometry. Parallel testing with MoAbs to CD16, CD11b, and CD45 served as controls. The size of the granulocyte population reacting with 1B5 and 7D8 and the respective mean, median, and peak cell fluorescence intensities were analyzed. RESULTS: The expression of 7D8 and 1B5 was greater on granulocytes from female donors. The expression of 7D8 fell in older women but not in men. There were no differences among the four racial groups in either the frequency of NB1 as determined by 1B5 or 7D8 or in the size of the population of granulocytes reacting with either antibody. When the fluorescence intensities of the antibody reactions were compared among groups, there were no differences in reactivity with 1B5. However, reactions with 7D8 were all greater in blacks. Comparison of the size of the antigen-positive granulocyte population, as determined by antibody reactivity, showed that only 30 donors differed by more than 10 percent. These discordant results were more likely to occur in whites than in blacks (18% vs. 4%, p<0.02). CONCLUSIONS: NB1 is composed of at least two epitopes as determined by serologic studies. The expression of both antigens is greater in females. The 7D8-reactive epitope appears to be more prevalent or more accessible on granulocytes of blacks. Variations in the expression of NB1 are more likely to occur in whites. The biochemical and molecular basis of these variations are not known.     

水痘-带状疱疹病毒糖蛋白E表达载体的构建

目的 构建水痘-带状疱疹病毒（VZV）糖蛋白E的真核表达载体。方法用PCR方法扩增VZV糖蛋白E基因，并将其克隆到真核表达载体pcDNA3．1并用双酶切和测序方法鉴定。结果扩增的目的基因包括了全长的糖蛋白E基因，长度约1．9kb。并且成功构建了糖蛋白E基因重组表达载体。结论构建的重组表达载体基因序列正确，为进一步研究此蛋白的免疫原性打下了基础。     

刚地弓形虫RH株主要表面抗原1的基因克隆及原核表达

目的 从刚地弓形虫RH株基因组DNA中扩增出其主要表面抗原1(SAG1)全长的基因编码序列，构建重组原核表达载体并在大肠埃希菌中进行表达，为进一步研制弓形虫病免疫诊断试剂盒打下基础。方法 根据弓形虫RH株主要表面抗原SAG1的cDNA序列设计一对引物，利用聚合酶链反应(PCR)技术从RH株弓形虫基因组中扩增出SAGl的全长基因，将其克隆到载体pGEX-4T-3中，并通过基因测序加以证实；重新设计引物将其亚克隆至原核表达载体pET-32c中，在大肠埃希菌中经IPTG诱导表达。结果 从刚地弓形虫RH株基因组中成功扩增到1030bp的全长SAG1基因，该基因在原核系统中经诱导表达出一分子量约37000大小的融合蛋白，表达产物以包涵体的形式存在。结论 SAG1是弓形虫主要的表面抗原，原核表达的sAG1融合蛋白在弓形虫病的免疫诊断中具有明显的潜在应用价值。     

结核分枝杆菌ESAT6基因克隆与表达质粒构建

目的　克隆结核分枝杆菌 (MTB)分泌蛋白ESAT6基因 ,并构建重组表达质粒。方法　根据Genbank中ESAT6基因序列 ,针对其编码区合成引物 ,采用PCR方法从结核分枝杆菌H37Rv基因组DNA中扩增出ESAT6基因 ,并连接到T载体 ,然后定向克隆到原核表达质粒pGEX 4T 2 ,阳性克隆用酶切和DNA测序鉴定。结果　经双内切酶消化所切下的片段 ,大小与预计相符 ;测序结果证实基因序列正确 ,符合表达框架。结论　成功构建了重组原核表达质粒 pGEX ESAT6。     

中介素真核表达载体的构建及其在超声微泡介导下的肾脏表达

目的 构建大鼠中介素(IMD)基因真核表达载体IMD-pCDNA3.1(+),以超声微泡法介导其在大鼠体内肾脏局部高表达.方法 用HindⅢ和EcoRI双酶切IMD基因和目的载体pCDNA3.1(+),连接制备真核表达载体IMD-pCDNA3.1(+),命名为IMD-pCDNA3.1(+),并对重组表达载体进行酶切、测序鉴定.18只雄性Wistar大鼠随机分为未转染组、空质粒转染组以及IMD基因转染组,每组6只,采用超声微泡介导技术转染大鼠肾脏.RT-PCR、Western blotting检测IMD表达.结果 经限制性酶切鉴定及测序分析证实IMD-pCDNA3.1(+)载体序列正确;3组大鼠肝、肾功能差异均无统计学意义(P均＞0.05);实验各组均未见明显肾小球及肾小管、间质损害;半定量RT-PCR、Western blotting检测显示:IMD转基因组肾组织IMD mRNA及其蛋白表达较空质粒转染组和未转染组明显上调.结论 IMD-pCDNA3.1(+)表达载体构建成功,并在大鼠肾脏内成功表达.

Abstract：

Objective To construct eukaryotic expression vector encoding rat IMD gene and deliver it into rat renal tissue via ultrasound-mircobubbles. Methods IMD gene was inserted into pCDNA3.1 ( + )between Hind Ⅲ and EcoRI enzyme sites. The recombinant plasmid designated as IMD-pCDNA 3.1 wasconfirmed by restrictive enzyme digestion and sequencing. Eighteen male Wistar rats were randomized into 3groups, which were treated with no transfection, empty vector transfection and IMD transfection, respectively, in renal tissue via ultrasound-microbubbles. RT-PCR and Western blotting were applied to detect the expression level of IMD. Results Enzyme- digestion and sequencing data showed that IMD-pCDNA 3.1 was correctly constructed. The differences in ALT, AST, BUN and SCr were not significant; No obvious damage in the glomerular, tubular and interstitial was observed in all the treated groups;Compared with non-transfection group and empty vector-transfection group, IMD mRNA and protein expression in IMD transgenic renal tissue were significantly increased. Conclusion IMD-pCDNA 3.1 expression vector was successfully constructed and well expressed in rat kidney.     

AAV serotype-1 mediates early onset of gene expression in mouse hearts and results in better therapeutic effect

Adeno-associated viral vectors (AAV) are attractive tool for gene therapy for coronary artery disease. However, gene expression in myocardium mediated by AAV serotype 2 (AAV2) does not peak until 4-6 weeks after gene transfer. This delayed gene expression may reduce its therapeutic potential for acute cardiac infarction. To determine whether earlier gene expression and better therapeutic effect could be achieved using a different serotype, CMV promoter driving the EPO gene (AAV-EPO) was packaged into AAV serotypes 1-5 capsids and injected into mouse myocardium. EPO expression was studied by measuring the hematocrits and EPO mRNA. After we found that AAV1 mediates the highest gene expression after 4 days of gene transduction, AAV-LacZ (CMV promoter driving LacZ gene expression) and MLCVEGF (hypoxia-inducible and cardiac-specific VEGF expression) were packaged into AAV1 and 2 capsids. LacZ expression was detected in AAV1-LacZ but not in AAV2-LacZ-injected hearts 1 day after vector injection. Compared to AAV2-MLCVEGF that mediated no significant VEGF expression, AAV1-MLCVEGF mediated 13.7-fold induction of VEGF expression in ischemic hearts 4 days after gene transduction and resulted in more neovasculatures, better cardiac function and less myocardial fibrosis. Thus, AAV1 mediates earlier and higher transgene expression in myocardium and better therapeutic effects.     

Lack of HLA class I antigen expression by cultured melanoma cells FO-1 due to a defect in B2m gene expression

The melanoma cell line FO-1 does not express HLA class I antigens and does not acquire them on the cell surface after incubation with IFN-gamma. Immunochemical studies showed that FO-1 cells synthesize HLA class I heavy chain, but do not synthesize beta 2-microglobulin (beta 2-mu). The latter abnormality is associated with lack of beta 2-mu mRNA which remains undetectable in FO-1 cells incubated with IFN-gamma. The defect was identified as a genetic lesion in the B2m gene, since DNA hybridization analysis detected a deletion of the first exon of the 5'-flanking region, and of a segment of the first intron of the B2m gene. HLA class I antigen expression was reconstituted on melanoma cells FO-1 after transfection with the wild-type mouse B2m gene, thereby confirming the abnormality of the endogenous B2m gene. The defect identified in FO-1 cells is distinct from that underlying the lack of HLA class I antigen expression by lymphoblastoid cells Daudi, but is remarkably similar to that causing lack of H-2 class I antigen expression by mouse lymphoblastoid cells R1 (TL-). These results suggest that genetic recombination in the 5' region of the B2m gene is a recurrent mechanism in B2m gene defects. In addition to contributing to our understanding of molecular abnormalities in HLA class I antigen expression by melanoma cells, FO-1 cells represent a useful model for analyzing the role of HLA class I antigens in the biology of melanoma cells and in their interaction with cells of the immune system.     

Immunological similarity of NCA (non-specific cross-reacting antigen) in feces with alpha 1-acid glycoprotein

We have recently suggested that carcinoembryonic antigen (CEA) may contain alpha 1-acid glycoprotein (AG) antigenic determinant. In the present work we examined a protein with CEA-like activity in the feces of healthy subjects (NCA) for immunological cross-reactivity with AG. When the perchloric acid extract of feces was fractionated on a Sephadex G-200, two fractions (large and small molecular weight) were obtained. The large molecular weight fraction had higher CEA activity than the small one. The perchloric acid (PCA) extract of feces was subjected to affinity chromatography using anti-CEA bound to Sepharose, and the bound protein was labelled with 125I, and then fractionated on a Sephadex G-200 column. Two radioactive peaks, Peak 1 corresponding to an approximate Mr of 180 000 and Peak 2, corresponding to an approximate Mr of 60 000 were found. Both peaks showed immunoreactivity with either anti-CEA or anti-AG. This experiment suggests the presence of two kinds of CEA-reactive proteins in feces: one which may be a big protein with immunological similarity to AG and a second which appears to be a hydrolysed fragment of this protein.