人类白细胞抗原-G突变体cDNA克隆及在K562细胞上的表达

目的：克隆人类白细胞抗原-G(Human leukocyte antligen-G，HLA-G)突变体cDNA，并使其在HLA-I类阴性的K562细胞上获得稳定表达，为研究配-受体之间的识别机制奠定基础。方法：用RT-PCR方法从人子宫蜕膜组织扩增出HLA-GcDNA，得到全长HLA-GPCR产物后，用桥式PCR方法进行定点点突变，将突变的目的基因亚克隆于逆转录，将突变的目的基因亚克隆于逆转录mG-pLNCX表达载体，采用感染的方法将重组质粒转入K562细胞，最后经G418筛选及有限稀释，利用单克隆抗体W6/32进行FACS及mRNA检测，观察HLA-G突变体在靶细胞表面的表达。结果：HLA-G突变体分子在经mG-pLNCX转染的靶细胞表面获得稳定高表达。结论：成功构建了mG-pLNCX表达载体，并使HLA-G突变体分子在HLA-I类阴性的靶细胞K562细胞上获得稳定表达。     

人白细胞介素13cDNA克隆及序列分析

人白细胞介素１３ｃＤＮＡ克隆及序列分析范灵芝，杨新科，段淑敏，刘彦仿，侯云德白细胞介素１３（Ｉｎｔｅｒｌｅｕｋｉｎ－１３，ＩＬ－１３）是近年来发现的一种新的免疫调节介质［１］，它主要由激活的Ｔ淋巴细胞产生，具有调节单核细胞及Ｂ淋巴细胞的功能，在病毒免...     

中国人IL—18结构蛋白cDNA的克隆和序列分析

目的克隆人白介素 - 18结合蛋白 (h IL- 18BP)的 c DNA,以研究其结构与功能的关系。方法从中国汉族人的胎盘组织中提取总 RNA,以 RT- PCR方法扩增出全长 5 85个 bp的 IL- 18BP c DNA,将其与表达载体 pc DNA3连接 ,转化大肠杆菌 DH5 α,建立了 h IL- 18BP的 c DNA克隆。结果序列分析表明 ,与国外文献报道的人 IL- 18BP的 c DNA序列完全一致。结论 h IL- 18BP已成功地得到克隆。     

人单核细胞趋化蛋白—1（MCP—1）cDNA的克隆和序列测定

人单核细胞趋化蛋白－１（ＭＣＰ－１）是由多种细胞产生的一种趋化因子。它不但能趋化单核细胞还能激活单核细胞，在机体防御。炎症恢复和抗肿瘤等方面起重要作用。本文利用ＲＴ－ＰＣＲ方法克隆了人ＭＣＰ－１ｃＤＮＡ，经核鞋酸序列测定，除第１２位密码子为ＴＧＴ与国外报道的ＴＧＣ不同，但编码相同的氨基酸半胱氨酸外，其余序列完全相同。     

藏猪白细胞介素4基因Cdna的克隆及序列分析

目的：克隆藏猪白细胞介素4基因cDNA。方法：从体外ConA刺激70小时的藏猪外周血淋巴细胞中提取总RNA，应用RT-PCR技术扩增，pMD-T载体连接，常规转化后，进行酶切及序列测定进行鉴定。结果：研究表明克隆得到的IL-4基因cDNA与成华猪的IL-4同源性达到99％，与长白杂交猪的同源率为98％。结论：从藏猪外周血淋巴细胞中成功地分离到IL-4基因。     

可溶性人白细胞抗原G与胚胎着床相关性研究进展

提高体外受精-胚胎移植技术的临床妊娠率,获得高质量的胚胎是胚胎学家为之奋斗的目标。目前评价胚胎质量普遍应用的是形态学观察,尚不能满足准确预测胚胎发育潜能的需要。s HLA-G因其在母-胎界面的独特分布和母-胎免疫耐受中所发挥的重要作用而引起关注,但对于胚胎培养液中s HLA-G能否作为胚胎质量的评价参数,反映胚胎的种植潜能,目前尚无定论。本文综述了国内外近年来胚胎培养液中s HLA-G与胚胎发育以及IVF-ET临床妊娠率的关系研究,努力为临床优选胚胎提供一种有效的非侵入性检测途径,提高妊娠率。     

人白细胞介素10（hIL—10）基因的克隆和序列测定

应用逆转录多聚酶链式反应（ＲＴ－ＰＣＲ）技术，自行设计并合成一对引物，成功地从活化的正常人外周血单个核细胞中扩增出ｈＩＬ－１０ｃＤＮＡ基因。并定向插入ｐＵＣ１８载体，经ＤＮＡ序列测定最终确定为ｈＩＬ－１０ｃＤＮＡ基因，这将为今后ｈＩＬ－１０基因探针的制备和基因表达，进一步在基因水平上探讨ｈＩＬ－１０的生物学功能提供了物质基础。     

中国4株丁型肝炎病毒抗原编码区基因的cDNA克隆与…

从我国四川、广西、河南的丁型肝炎、病毒抗原（ＨＤＡｇ）或抗体阳性的ＨｂｓＡｇ携带者中，筛选出４株ＨＤＶ ＲＮＡ阳性标本，经逆转录和聚合酶反应（ＲＴ－ＰＣＲ）后，获得包含ＨＤＶ抗原编码区的ｃＤＮＡ片段，并对其进行克隆与序列测定。经计算机分析比较表明：四川、广西株与美国－１株同源性最高，分别为９９．３％、９９．０％；而河南－１、河南－２株与台湾株的同源性较高，分别为９４．３％、９２．１％；上述４株与日     

可溶性人类白细胞抗原G与重度子痫前期的相关性研究

目的：在重度子痫前期患者血浆中的含量水平,探讨s HLA-G在早发型及晚发型重度子痫前期患者血浆中表达的差异性。方法：选择南京医科大学附属南京医院2013年07月至2014年04月间收治的6例早发型重度子痫前期患者（早发型组）、18例晚发型重度子痫前期患者（晚发型组）的临床资料,比较早发型组及晚发型组患者的一般临床情况;应用免疫组织化学染色法（enzyme linked immunosorbent assay,ELISA）,检测6例早发型组,18例晚发型组及32例正常足月妊娠者（正常对照组）的血清中s HLA-G的表达,并比较三组s HLA-G含量的差异。结果：1）早发型组与晚发型组的发病孕周,分娩孕周及期待治疗天数差异均有统计学意义（P〈0.05）,而年龄差异无统计学意义（P〉0.05）;2）早发型组与晚发型组的收缩压及舒张压差异均有统计学意义（P〈0.05）;3）早发型组中s HL A-G含量均值为（4.284±1.932）ng/m L,晚发型组中s HL A-G含量均值为（7.036±1.623）ng/m L,正常孕妇组中s HLA-G含量均值为（10.399±3.168）ng/m L。正常对照组s HLA-G含量高于重度子痫前期组（P〈0.01）,且早发型组低于晚发型组（P〈0.05）。     

Natural killer cell functional activity suppression by intravenous immunoglobulin, intralipid and soluble human leukocyte antigen-G

PROBLEM: The purpose of this study was to compare the ability of intravenous immunoglobulin (IVIg), intralipid and soluble human leukocyte antigen (sHLA)-G to suppress natural killer (NK) cell cytotoxicity in an in vitro assay. METHOD OF STUDY: Blood samples taken from 275 women experiencing reproductive failure were analyzed for NK cytotoxicity and the suppression of NK cytotoxicity by IVIg 4 and 2 mg/mL (n = 275), intralipid 18 and 9 mg/mL (n = 275) and sHLA-G 70 and 35 ng/mL (n = 50) using immunofluorescent labeled K562 cells as targets and flow cytometry. RESULTS: Natural killer cytotoxicity was suppressed in all samples. Among patients with normal NK cell activity, IVIg suppressed NK cytotoxicity by 44.9 +/- 8.1%, intralipid suppressed NK killing by 45.2 +/- 8.3% and sHLA-G suppressed by 49.0 +/- 9.2%. When specimens with abnormal NK activity were observed for suppression of cytotoxicity, IVIg suppressed by 38.9 +/- 5.4%, intralipid suppressed by 39.8 +/- 6.2% and sHLA-G suppressed by 39.9 +/- 5.0%. CONCLUSION: Intravenous immunoglobulin, intralipid and sHLA-G suppressed NK cell cytotoxicity with equal efficacy in an in vitro assay.     

Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines

Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.     

Diagnostic significance of soluble human leukocyte antigen-G for gastric cancer

Human leukocyte antigen-G (HLA-G) is a novel tumor marker. Increased level of soluble HLA-G (sHLA-G) in various tumor types has been reported. However, the potential diagnostic value of sHLA-G with other tumor markers in gastric cancer (GC) diagnosis is yet to be explored. In this study, plasma level of sHLA-G was measured in 81 GC patients, 53 benign gastric disease patients and 77 normal controls by ELISA. The serum levels of alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 19-9 (CA19-9) and cancer antigen 72-4 (CA72-4) were also determined. Data showed that plasma level of sHLA-G in GC was dramatically increased compared with normal controls and benign gastric disease patients (both p < 0.001). The AUC for sHLA-G was 0.730 (p < 0.001), superior to serum AFP, CEA, CA125, CA19-9 and CA72-4. After evaluating three cut-offs of sHLA-G, we concluded sHLA-G (cut-off at 128 U/ml) plus CA125 in two-biomarker panel test and CA125 plus CA199 plus sHLA-G or CA125 plus CA724 plus sHLA-G in three-biomarker panel test were better choices for GC discrimination. Our findings indicated that sHLA-G was a potential biomarker for GC diagnosis and the combination of sHLA-G with CA125, CA19-9 and CA72-4 can improve the clinical screening and diagnosis for GC.     

主动免疫治疗对URSA患者外周血sHLA—G的表达影响的研究

目的检测URSA患者经主动免疫治疗前后外周血中sHLA—G的水平表达,为主动免疫治疗对URSA患者疗效评价提供一定的理论及方法学依据。方法将研究对象分为四组,URSA组、URSA主动免疫治疗组、正常早孕组及正常未孕组。用ELISA法定量检测样本中sHLA—G的表达情况,RT—PCR半定量检测外周血中sHLA—G的表达情况。结果（1）正常早孕组血清中sHLA—G的表达水平显著高于原因不明性流产组以及正常未孕组（P〈0．05）;URSA患者主动免疫治疗组sHLA—G的表达水平高于原因不明性流产组以及正常未孕组（P〈0．05）,与正常早孕组相比无统计学意义（P〉0．05）;（2）RT—PCR半定量检测显示URSA组及正常未孕组sHLA—G表达均低于正常早孕组及治疗组（P〈0．05）。结论淋巴细胞主动免疫治疗可以提高外周血中sHLA—G的表达,主动免疫治疗能够有效治疗URSA妊娠后的流产患者。     

Accuracy of soluble human leukocyte antigen-G for predicting pregnancy among women undergoing infertility treatment: meta-analysis

BACKGROUND: There have been concerns about validity and accuracy of themeasurement of sHLA-G in embryo culture supernatants. In thissystematic review, we quantified the diagnostic accuracy ofsHLA-G for predicting the ability to achieve clinical pregnancyin women who are undergoing infertility treatment. METHODS: Medline and Embase were searched up to 7 September 2007, forfull English and non-English articles concerning cohort studiesevaluating sHLA-G in embryo culture for predicting clinicalpregnancy in women undergoing IVF and ICSI. RESULTS: Eleven studies including 1813 patients met our inclusion criteria.In the individual studies, sensitivity ranged from 0.01 to 0.97,specificity from 0.18 to 0.98, the positive likelihood ratiofrom 0.34 to 3.21 and the negative likelihood ratio from 0.08to 1.01. These values were highly heterogeneous with, in eachcase, I² values of >75%, and P-values for the Q statisticof <0.001, arguing against generating a pooled estimate forthese diagnostic test properties. The diagnostic odds ratios(DORs) ranged from 0.92 to 24.82 in the individual studies withan I² value of 49% indicating moderate heterogeneity. Therefore,the meta-analysis combined the logs of the DORs, which are derivedfrom sensitivity and specificity. A random-effects model yieldeda summary DOR of 4.38 (95% CI, 2.93–6.55), consistentwith modest diagnostic accuracy. Interestingly, an a prioridefined subgroup analysis restricted to six studies with goodquality embryos showed a better diagnostic performance witha DOR of 12.67 (95% CI, 3.66–43.80) to predict the abilityto achieve clinical pregnancy in women undergoing infertilitytreatment. CONCLUSIONS: Further research is needed with single-embryoculture, single-embryo transfer and highly sensitive detectiontechniques to determine the potential application of measuringsHLA-G in culture supernatant.     

人白细胞介素15（hIL—15）的基因克隆和序列测定

ＩＬ１５是一种促进Ｔ细胞、Ｂ细胞和ＮＫ细胞生长和分化的细胞因子。本文用ＲＴＰＣＲ方法，自行设计并合成两对引物，成功地从成人脾脏中扩增出ｈＩＬ１５ｃＤＮＡ，并定向克隆入ｐＵＣ１９载体中。经序列测定证实，为ｈＩＬ１５成熟肽ｃＤＮＡ基因。ｈＩＬ１５基因的获得，为进一步在大肠杆菌中高效表达有活性的重组ＩＬ１５，更深入地研究其作用机理，以及临床应用奠定了基础。     

MAGE—1基因在人食管癌中的表达及基因克隆

目的 研究MAGE-1基因在人食管癌中的表达,分析MAGE-1抗原能否作为食管癌的治疗性候选疫苗。方法 采用RT-PCR方法检测了MAGE-1基因在30例食管癌组织和相应癌旁正常组织中的表达;并从食管癌组织中扩增了MAGE-1cDNA全长序列,经酶切后与pET-30a( )质粒载体连接,经初步酶切鉴定后,进行DNA序列分析。获得克隆基因的核苷酸序列。结果 MAGE-1基因在食管癌组织中的表达率为43%（13/30）,在30例相应癌旁正常组织中未见表达;成功地克隆了MAGE-1cDNA全长序列。结论 由于MAGE-1基因在食管癌中高频率表达以及MACE-1抗原具有HLA-I类分子限制性CTL识别表位,因此,MAGE-1抗原可以作为食管癌免疫治疗的候选疫苗。     

Elevated levels of soluble non-classical major histocompatibility class I molecule human leucocyte antigen (HLA)-G in the blood of HIV-infected patients with or without visceral leishmaniasis

The non-classical class I major histocompatibility complex molecules human leucocyte antigen (HLA)-G have been shown to play a role in HIV persistence, but no data are available on the expression of the soluble forms HLA-G5 and sHLA-G1 in HIV-infected patients with and without opportunistic infections. The soluble HLA-G isoform was measured with an enzyme-linked immunosorbent assay (ELISA) method in plasma from 94 subjects: 31 HIV-1-seropositive, 17 with visceral leishmaniasis (VL), seven with both VL and HIV-1 infection and 39 healthy HIV-seronegative subjects. Between groups, the frequency of sHLA-G positivity was statistically different: 81% of HIV-infected patients were positive, as were 57% of HIV-Leishmania infantum co-infected patients, 35% of HIV-seronegative patients with VL and 3% of healthy controls. Levels of the soluble forms of the immunomodulatory molecules HLA-G are elevated during HIV infection. In HIV-Leishmania co-infected patients, sHLA-G secretion could contribute to the tolerogenic environment and to Leishmania immune evasion.