中华实验和临床感染病杂志（电子版）
中華實驗和臨床感染病雜誌（電子版）
중화실험화림상감염병잡지（전자판）
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL INFECTIOUS DISEASES(ELECTRONIC VERSION)
2013年
3期
325-331
,共7页
张仁雯%康艳芳%乔雍%郝晓花%常路丝%李红敏%任慧%张晓静%孟雪%李兴旺%魏红山
張仁雯%康豔芳%喬雍%郝曉花%常路絲%李紅敏%任慧%張曉靜%孟雪%李興旺%魏紅山
장인문%강염방%교옹%학효화%상로사%리홍민%임혜%장효정%맹설%리흥왕%위홍산
FAM172A基因%多克隆抗体%肝细胞%细胞增殖
FAM172A基因%多剋隆抗體%肝細胞%細胞增殖
FAM172A기인%다극륭항체%간세포%세포증식
FAM172A gene%Polyclonal antibody%Hepatocyte%Cell proliferation
目的体外克隆人类基因FAM172A，构建其原核表达载体并诱导其重组蛋白的表达，制备兔抗FAM172A重组蛋白的多克隆抗体，观察其在不同细胞系的表达情况。观察FAM172A-1和FAM172A-3蛋白对L02细胞增殖的影响。方法利用反转录聚合酶链反应（RT-PCR）及PCR技术，构建原核表达质粒pET-32a（＋）-FAM172A-1和pET-32a（＋）-FAM172A-3。诱导该基因不同异构体重组蛋白的表达，并通过蛋白质免疫印迹（Western blot）技术进行鉴定。纯化后的重组蛋白FAM172A-1和FAM172A-3与肝细胞L02细胞共孵育，观察不同浓度的重组蛋白对细胞增殖的影响。利用纯化后的FAM172A（异构体3）重组蛋白免疫大耳白兔，获得抗FAM172A-3蛋白的多克隆抗体，利用酶联免疫吸附法（ELISA）以及Western blot技术对获得的多克隆抗体进行效价分析及特异性检测。结果成功扩增获得FAM172A（异构体1、3）的基因片段，测序结果与GenBank已公开的基因序列一致；成功表达FAM172A（异构体1、3）的重组蛋白，经过Western blot鉴定正确；纯化后的重组蛋白FAM172A （异构体1、3）与L02细胞共孵育结果发现，两者对L02细胞在一定浓度范围内均有促进细胞增殖的作用。所制备的兔抗人FAM172A-3重组蛋白的多克隆抗体，ELISA检测显示其效价可达1︰1280000， Western blot检测证实该多克隆抗体的特异性良好；Western blot分析显示，该蛋白在肝脏间质细胞和实质细胞均有一定程度的表达。结论 FAM172A蛋白在肝实质细胞及肝间质细胞均表达，且其重组蛋白可以促进L02细胞增殖，推测该基因可能与肝细胞损伤、肝纤维化以及肝细胞再生等发生机制有关。
目的體外剋隆人類基因FAM172A，構建其原覈錶達載體併誘導其重組蛋白的錶達，製備兔抗FAM172A重組蛋白的多剋隆抗體，觀察其在不同細胞繫的錶達情況。觀察FAM172A-1和FAM172A-3蛋白對L02細胞增殖的影響。方法利用反轉錄聚閤酶鏈反應（RT-PCR）及PCR技術，構建原覈錶達質粒pET-32a（＋）-FAM172A-1和pET-32a（＋）-FAM172A-3。誘導該基因不同異構體重組蛋白的錶達，併通過蛋白質免疫印跡（Western blot）技術進行鑒定。純化後的重組蛋白FAM172A-1和FAM172A-3與肝細胞L02細胞共孵育，觀察不同濃度的重組蛋白對細胞增殖的影響。利用純化後的FAM172A（異構體3）重組蛋白免疫大耳白兔，穫得抗FAM172A-3蛋白的多剋隆抗體，利用酶聯免疫吸附法（ELISA）以及Western blot技術對穫得的多剋隆抗體進行效價分析及特異性檢測。結果成功擴增穫得FAM172A（異構體1、3）的基因片段，測序結果與GenBank已公開的基因序列一緻；成功錶達FAM172A（異構體1、3）的重組蛋白，經過Western blot鑒定正確；純化後的重組蛋白FAM172A （異構體1、3）與L02細胞共孵育結果髮現，兩者對L02細胞在一定濃度範圍內均有促進細胞增殖的作用。所製備的兔抗人FAM172A-3重組蛋白的多剋隆抗體，ELISA檢測顯示其效價可達1︰1280000， Western blot檢測證實該多剋隆抗體的特異性良好；Western blot分析顯示，該蛋白在肝髒間質細胞和實質細胞均有一定程度的錶達。結論 FAM172A蛋白在肝實質細胞及肝間質細胞均錶達，且其重組蛋白可以促進L02細胞增殖，推測該基因可能與肝細胞損傷、肝纖維化以及肝細胞再生等髮生機製有關。
목적체외극륭인류기인FAM172A，구건기원핵표체재체병유도기중조단백적표체，제비토항FAM172A중조단백적다극륭항체，관찰기재불동세포계적표체정황。관찰FAM172A-1화FAM172A-3단백대L02세포증식적영향。방법이용반전록취합매련반응（RT-PCR）급PCR기술，구건원핵표체질립pET-32a（＋）-FAM172A-1화pET-32a（＋）-FAM172A-3。유도해기인불동이구체중조단백적표체，병통과단백질면역인적（Western blot）기술진행감정。순화후적중조단백FAM172A-1화FAM172A-3여간세포L02세포공부육，관찰불동농도적중조단백대세포증식적영향。이용순화후적FAM172A（이구체3）중조단백면역대이백토，획득항FAM172A-3단백적다극륭항체，이용매련면역흡부법（ELISA）이급Western blot기술대획득적다극륭항체진행효개분석급특이성검측。결과성공확증획득FAM172A（이구체1、3）적기인편단，측서결과여GenBank이공개적기인서렬일치；성공표체FAM172A（이구체1、3）적중조단백，경과Western blot감정정학；순화후적중조단백FAM172A （이구체1、3）여L02세포공부육결과발현，량자대L02세포재일정농도범위내균유촉진세포증식적작용。소제비적토항인FAM172A-3중조단백적다극륭항체，ELISA검측현시기효개가체1︰1280000， Western blot검측증실해다극륭항체적특이성량호；Western blot분석현시，해단백재간장간질세포화실질세포균유일정정도적표체。결론 FAM172A단백재간실질세포급간간질세포균표체，차기중조단백가이촉진L02세포증식，추측해기인가능여간세포손상、간섬유화이급간세포재생등발생궤제유관。
Objective To clone human gene FAM172A in vitro and to establish prokaryotic expression vector of human gene FAM172A. To induce the expression of their recombinant proteins and rabbit anti-FAM172A-3 protein polyclonal antibody was prepared. To observe the expression of FAM172A (isomer 1, 3) in different cell lines and the effect of FAM172A protein on proliferation of cultured L02 cells. Methods With reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR), the gene fragments were cloned into prokaryotic expression vector pET-32a (+). The expression of gene recombinant proteins was induced and analyzed by Western blot. Puriifed recombinant protein and L02 cells were incubated, and the effect of proliferation of cultured L02 cells was analyzed. The FAM172A-3 puriifed recombinant protein was used to immunize the big ear rabbits to obtain polyclonal antibody. The potency and speciifcity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results The recombinant protein of FAM172A were highly expressed. The recombinant FAM172A-1 and FAM172A-3 gene had the same DNA sequence with GenBank and the same molecular weight with prediction accessed by Western blot. ELISA analysis indicated that the titer of polyclonal antibody could achieve at 1︰1 280 000. The high speciifcity of polyclonal antibody was conifrmed by Western blot. Puriifed recombinant protein and L02 cells were incubated and the recombinant protein of FAM172A could promote L02 cells proliferation within a certain concentration range. Conclusions FAM172A (isomer 1, 3) expressed in hepatocytes and liver interstitial cells, which indicated it might be associated with hepatocellular injury and the mechanism of liver ifbrosis. The recombinant protein of FAM172A could promote L02 cells proliferation within a certain concentration range, which might be associated with liver cell regeneration.
