Prognosis value of heat-shock proteins in esophageal and esophagogastric cancer:A systematic review and meta-analysis

BACKGROUND Heat shock proteins(HSPs)are molecular chaperones that play an important role in cellular protection against stress events and have been reported to be overex-pressed in many cancers.The prognostic significance of HSPs and their regulatory factors,such as heat shock factor 1(HSF1)and CHIP,are poorly understood.AIM To investigate the relationship between HSP expression and prognosis in esophageal and esophagogastric cancer.METHODS A systematic review was conducted in accordance with PRISMA recommend-ations(PROSPERO:CRD42022370653),on Embase,PubMed,Cochrane,and LILACS.Cohort,case-control,and cross-sectional studies of patients with eso-phagus or esophagogastric cancer were included.HSP-positive patients were compared with HSP-negative,and the endpoints analyzed were lymph node metastasis,tumor depth,distant metastasis,and overall survival(OS).HSPs were stratified according to the HSP family,and the summary risk difference(RD)was calculated using a random-effect model.RESULTS The final selection comprised 27 studies,including esophageal squamous cell carcinoma(21),esophagogastric adenocarcinoma(5),and mixed neoplasms(1).The pooled sample size was 3465 patients.HSP40 and 60 were associated with a higher 3-year OS[HSP40:RD=0.22;95%confidence interval(CI):0.09-0.35;HSP60:RD=0.33;95%CI:0.17-0.50],while HSF1 was associated with a poor 3-year OS(RD=-0.22;95%CI:-0.32 to-0.12).The other HSP families were not associated with long-term survival.HSF1 was associated with a higher probability of lymph node metastasis(RD=-0.16;95%CI:-0.29 to-0.04).HSP40 was associated with a lower probability of lymph node dissemination(RD=0.18;95%CI:0.03-0.33).The expression of other HSP families was not significantly related to tumor depth and lymph node or distant metastasis.CONCLUSION The expression levels of certain families of HSP,such as HSP40 and 60 and HSF1,are associated with long-term survival and lymph node dissemination in patients with esophageal and esophagogastric cancer.

Preparation of Monoclonal Antibody against P65 Protein of Mycoplasma hyopneumoniae

P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.

Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism

<abstract>Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.

Effects of moxibustion on heat-shock protein 70 expression in the spinal cord and colonic mucosa in a rat model of ulcerative colitis

Pathological changes in the colon are closely associated with the spinal cord, and innervation of spinal cord can regulate cellular functions. Our previous studies verified that moxibustion protects and restores the colonic mucosa, but the mechanisms of action remain unknown. The present study observed the effects of moxibustion and salicylazosulfapyridine on expression of heat-shock protein 70 （HSP70） and its mRNA in the spinal cord and colonic mucosa of ulcerative colitis rats. Results demonstrated that moxibustion and salicylazosulfapyridine increased HSP70 mRNA expression in the spinal cord and colonic mucosa of ulcerative colitis rats. The decreased transcriptional activity of HSP70 in the spinal cord and colonic mucosa might participate in damage to the colonic mucosa in ulcerative colitis rats. Moxibustion exerted protective effects on colonic mucosa by up-regulating HSP70 transcriptional activity in the spinal cord and colonic mucosa.

Recombinant Mycobacterium smegmatis expressing Hsp65-hIL-2 fusion protein and its influence on lymphocyte function in mice

Objective:To coastruct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2(IL-2) fusion protein(rMS-Hsp65/IL-2) and to explore the effect of this construct on lymphocyte function in mice.Methods:The fusion gene encoding Hsp65-hIL-2 was cloned into shuttle vector pSMT3.The recombinant plasmid pSMT3- Hsp65-hIL-2 was transferred to Mycobacterium smegmatis by electroporation.Positive clones were selected by hygromycin and identified by PCR.The expression of fusion protein Hsp65- hIL-2 was verified using indirect immunofluorescence staining.Mice were immunized for two times by subcutaneously injection with 1×10~6 CFU rMS-Hsp65/IL-2 at a three-week interval.Two weeks after the second immunization,mice were sacrificed and the serum samples were collected for determination of anli-Hsp65 specific IgG.Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay.IFN-γand IL-2 in the medium of the treated cells were also determined by ELISA.Results:Successful construction of rMS-Hsp65/IL-2 was verified by PCR and immunofluorescence staining.Compared to the splenic lymphocytes isolated from mice immunized with Bacille Calmette-Guerin or mice immunized with Mycobacterium smegmatis alone,the splenic lymphocytes isolated from mice immunized with rMS-Hsp65/IL-2 showed a marked increase in the proliferation of lymphocytes, together with an increased production of important cytokines such as IFN- 7 and IL-2. Conclusions:rMS-Hsp65/IL-2 markedly enhances lymphocyte function.Therefore,the fusion protein generated by rMS-Hsp65/lL-2 may be of potential value in generating an effective vaccine against tuberculosis.

Hepatitis C virus inhibitor synergism suggests multistepinteractions between heat-shock protein 90 and hepatitis Cvirus replication

AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the intracellular and extracellular(culture medium) levels of the components(RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A(Cs A) or interferon. Finally, we statistically examined the combined effect of radicicoland Cs A using the combination index(CI) and graphical representation proposed by Chou and Talalay.RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor(Cs A or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy(CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

结核分枝杆菌HSP65与IL-2融合蛋白的表达和纯化

目的获得融合表达的结核分枝杆菌热休克蛋白65与人白细胞介素2的重组蛋白,为进一步研究其对结核疫苗的作用奠定基础。方法用PCR的方法分别从H37Rv DNA和质粒pGEM-Teasy-IL-2中扩增目的基因片段hsp65和IL-2,将各目的片段克隆至pMD18-T载体中进行测序,将测序正确的目的基因片段分别经EcoRI和ClaI,ClaI和HindⅢ双酶切后亚克隆至原核表达载体pPro-EX HTa,转化大肠杆菌DH5α,挑选阳性克隆,经IPTG诱导后进行融合表达,并通过镍柱对融合蛋白进行纯化。结果PCR法扩增获得的各目的基因片段与GenBank报道的一致。构建的融合蛋白原核表达载体在大肠杆菌中表达后,经SDS-PAGE和Western-blot分析,在Mr 78000处有特异性的蛋白表达条带。用镍柱进行亲和层析纯化后得到了高纯度的HSP65-IL-2融合蛋白。结论成功的构建了结核分枝杆菌HSP65与人IL-2的融合表达载体,并在大肠杆菌中获得高效表达,有望为结核的预防提供有效的疫苗。

热休克蛋白65及其融合蛋白Hsp65-6×P277的分离纯化和稳定性研究(英文)

来源于牛分枝杆菌的热休克蛋白65(Hsp65)在没有佐剂的情况下可作为载体分子将抗原表位递呈给免疫系统。热休克蛋白具有蛋白酶的催化活性,容易发生自溶而降解,这制约了基于Hsp65疫苗的研究。该研究中,建立了纯化Hsp65及其融合蛋白Hsp65-6×P277(P277为来源于人热休克蛋白60的肽段,线性重复6次)的方法。Hsp65与Hsp65-6×P277以可溶形式表达于大肠杆菌。在低温及添加EDTA的条件下经细胞裂解、硫酸铵沉淀及阴离子交换树脂等纯化方法,可得到电泳纯的目的蛋白。用纯化的融合蛋白Hsp65-6×P277免疫小鼠,可激发机体产生强烈的免疫应答,该融合蛋白有希望作为免佐剂的糖尿病疫苗加以进一步研究开发。

皮下免疫热休克蛋白65对高密度脂蛋白抗炎抗氧化功能的影响

目的观察不同剂量热休克蛋白65对载脂蛋白E基因敲除小鼠高密度脂蛋白抗炎抗氧化功能的影响。方法 8周龄载脂蛋白E基因敲除小鼠随机分为磷酸盐缓冲液对照组、5μg热休克蛋白65组及25μg热休克蛋白65组,分别于第3、6周进行皮下免疫。第16周取标本,分别检测血脂水平、血清屏氧酶1活性、髓过氧化物酶活性、高密度脂蛋白炎症指数及炎症因子白细胞介素10和干扰素γ含量。结果随着热休克蛋白65剂量的增加,血清高密度脂蛋白胆固醇水平和白细胞介素10表达量逐渐下降,屏氧酶1活性逐渐降低,高密度脂蛋白炎症指数、髓过氧化物酶活性和干扰素γ表达量则逐渐升高。与对照组比较,25μg热休克蛋白65组血清高密度脂蛋白胆固醇水平和白细胞介素10表达量明显下降(P<0.01),屏氧酶1活性明显降低(P<0.05),高密度脂蛋白炎症指数、髓过氧化物酶活性、干扰素γ表达量则显著升高(P<0.01)。结论热休克蛋白65可引起炎症反应,损害高密度脂蛋白抗炎抗氧化功能,并且这种作用与其剂量有关。

结核杆菌热休克蛋白65“自杀性”DNA疫苗的构建及其免疫效应研究

目的构建表达结核杆菌热休克蛋白65(HSP65)的“自杀性”DNA疫苗。方法用聚合酶链反应,从我室构建的表达结核杆菌HSP65真核表达质粒中(pCHSP65),扩增出编码HSP65的基因,与“自杀性”DNA载体pSFV重组。经酶切、测序鉴定,间接免疫荧光(IF)证实其能表达HSP65后,将此“自杀性”DNA疫苗(pAHSP65)免疫小鼠。用ELISA、MTT法研究其免疫效应,并观察其对结核菌感染小鼠的保护作用。结果酶切、测序鉴定结果证实为结核杆菌HSP65基因,在BHK21细胞中经IF证实有HSP65表达。该疫苗接种能诱导小鼠产生特异性抗体,刺激淋巴细胞增殖,并能抵抗结核杆菌的感染。结论我们成功地构建了表达结核杆菌HSP65的“自杀性”DNA疫苗,该疫苗能诱导特异性免疫应答,并对结核菌感染的小鼠有保护作用。其免疫效应优于常规的HSP65DNA疫苗。

pIHsp65GM的构建及其对结核杆菌感染小鼠的保护

目的构建pIHsp65GM双顺反子真核表达质粒,并在真核细胞中表达,研究该基因疫苗的免疫原性及其对小鼠感染结核杆菌后的免疫保护效果。方法制备基因疫苗,将C57BL/6小鼠于胫前肌注射质粒DNA免疫,末次免疫后用h37rv强毒株经尾静脉攻击小鼠,计数肺和脾组织中结核杆菌的菌落数,对小鼠的部分肺和脾组织作病理切片,经HE染色观察组织病变的程度,测血清特异性IgG,MTT测定脾淋巴细胞特异性增殖指数,测小鼠脾细胞培养上清中IFN-γ的水平,乳酸脱氢酶(LDH)释放法测定免疫小鼠特异性细胞毒性T细胞(CTL)活性。结果 pIHsp65GM基因疫苗构建成功并且诱导小鼠特异性IgG的产生、脾淋巴细胞增殖以及IFN-γ的分泌(平均含量显著高于2个阴性对照组(P<0.001))。pIHsp65GM免疫组小鼠的脾和肺的平均结核菌载量分别低于两个阴性对照组的相应器官的结核菌载量(P<0.001),同时也显著高于BCG免疫对照组。结论成功构建和表达pIHsp65GM质粒,该基因疫苗对小鼠结核杆菌感染有一定的免疫保护效果。

HSP65-MUC1肿瘤疫苗对小鼠胰腺组织的分子模拟作用

目的:探讨重组抗肿瘤融合蛋白疫苗热激蛋白65-黏蛋白1(HSP65-MUC1)在小鼠体内通过分子模拟方式损伤小鼠胰腺的可能性。方法:21只C57BL/6小鼠随机分为PBS对照组、HSP65组和HSP65-MUC1组(每组7只),分别皮下注射PBS、HSP65和HSP65-MUC1,每周1次,给药3周。无菌分离小鼠脾细胞,利用流式细胞术检测特异性淋巴细胞的增殖情况,组织病理学观察小鼠胰腺的病理改变。结果:流式细胞术检测,与PBS对照组和HSP65组比较,HSP65-MUC1肿瘤疫苗组HSP65-MUC1特异性淋巴细胞升高了16.88%(P<0.001);HSP65特异性淋巴细胞升高了7.29%(P<0.01);与HSP60交叉识别的特异性淋巴细胞升高了5.79%(P<0.05)。3个免疫组小鼠胰腺组织HE染色病理均正常。结论:HSP65-MUC1肿瘤疫苗没有通过分子模拟诱导损伤小鼠胰腺。

鼻粘膜免疫融合蛋白Hsp65-6×p277预防NOD小鼠1型糖尿病的发生(英文)

目的提高多肽p277的免疫原性,从而提高其对自身免疫性糖尿病的预防作用。方法将p2776次重复与Hsp65融合置于pET28a中构建重组Hsp65-6×p277表达质粒。该重组质粒在大肠杆菌BL21中以高效可溶形式表达。依次通过细胞裂解、硫酸铵沉淀、双蒸水透析、DEAE纤维素52柱层析纯化获得目的蛋白。用纯化后的融合蛋白Hsp65-6×p277通过鼻腔给药方式,在不添加任何佐剂的情况下3次免疫4周龄雌性NOD小鼠。每月眼角取血,检测抗体和血糖浓度。结果初步药效学实验表明融合蛋白Hsp65-6×p277可抑制NOD小鼠中1型糖尿病的发生。结论融合蛋白Hsp65-6×p277有可能发展成为一种具有防治胰岛素依赖性糖尿病作用的疫苗。

溃疡性结肠炎发病新机制以及热休克蛋白65的作用

溃疡性结肠炎是一种肠道非特异慢性炎症疾病,被世界卫生组织列为难治疾病,目前尚无特效治疗手段.溃疡性结肠炎病因和发病机制尚不十分清楚,限制了治疗药物的开发.对肠道菌群、热休克蛋白家族在溃疡性结肠炎发病中的作用以及热休克蛋白65的潜在治疗作用,以期加深对溃疡性结肠炎发病机制认识,为寻找潜在治疗靶点及药物开发提供借鉴.

热休克蛋白65对小鼠溃疡性结肠炎的作用机制研究

目的探讨热休克蛋白65(HSP65)对小鼠溃疡性结肠炎(UC)的作用及机制。方法选择C57BL/6小鼠,应用葡聚糖硫酸钠法复制UC模型。6只正常小鼠为正常对照组(A组),48只UC小鼠随机分成模型对照组(B组)、低剂量组(C组)、中剂量组(D组)及高剂量组(E组),每组12只。A组和B组小鼠灌胃给予磷酸盐缓冲液(PBS),C组、D组、E组小鼠分别灌胃给予HSP65,剂量分别是0.5、2.5及5.0mg/kg体重。隔天灌胃1次,共7次,末次灌胃2d后,麻醉处死小鼠。计算小鼠疾病活动指数(DAI)。取小鼠结肠中段组织,HE染色镜检并计算组织病理评分。取小鼠血液、脾脏和肠系膜淋巴结,分离有核细胞后采用流式细胞术检测调节性T细胞(Treg)活化情况。结果 HSP65蛋白治疗UC小鼠14d,B、C、D及E组小鼠的DAI分别为(3.66±0.39)、(3.07±0.15)、(1.50±0.43)和(0.83±0.31),C组、D组、E组小鼠DAI变化趋势有差异(P<0.05);C组、D组和E组小鼠结肠组织病理评分均低于B组小鼠(P<0.05);E组小鼠结肠组织病理评分低于C组和D组(P<0.05),D组小鼠结肠组织病理评分低于C组(P<0.05);与未治疗的B组比较,C组、D组、E组小鼠肠系膜淋巴结中的Treg细胞比例均高于B组(P?<0.05)。结论 HSP65蛋白可以缓解小鼠溃疡性结肠炎,其机制可能是通过激活小鼠肠系膜淋巴结中的静止Treg细胞,使得活化Treg细胞比例增加,再通过活化Treg细胞发挥抑制肠道炎症反应作用。

PSI和CURB-65评分在评估医疗护理相关性肺炎病情严重程度中的应用

目的探讨肺炎严重度指数(PSI)和CURB-65评分及降钙素原(PCT)、C反应蛋白(CRP)在医疗护理相关性肺炎(HCAP)病情评估中的应用价值,以指导临床治疗。方法按照我国肺炎分类标准及PSI和CURB-65评分系统将92例HCAP患者分为高危组、中危组及低危组,分别比较PCT、CRP、白细胞(WBC)和中性粒细胞百分比(Neu)在3组间的差异性和相关性;构建ROC曲线分析PSI、CURB-65评分及血清PCT、CRP对住院HCAP患者死亡的预测价值。结果重症组HCAP患者PSI和CURB-65评分以及PCT、CRP、WBC和Neu水平显著高于非重症组HCAP患者(P<0.05);按PSI和CURB-65评分高危组HCAP患者血清PCT、CRP、WBC和Neu水平显著高于低危组(P<0.05),其中WBC和Neu%水平显著高于中危组。PSI和CURB-65评分与血清PCT及CRP水平呈正相关。入院时PSI评分>120分或CURB-65评分>2分为死亡的预测因素。结论 PSI和CURB-65评分与HCAP的病情严重程度具有一定的相关性,联合并监测血清PCT及CRP水平可以提高HCAP患者严重程度的预测准确性。

同位素^(65)Zn示踪法在研究膳食蛋白质与锌相互关系中的应用

本实验研究了不同来源膳食蛋白质及其摄取水平对大鼠锌吸收、分布、潴留、排泄及生物利用率的影响。结果表明：发芽大豆在各项指标上均优于普通大豆（Ｐ＜０．０１）；粪锌及粪￣（６５）Ｚｎ排出随膳食蛋白质水平的升高呈明显下降趋势，低蛋白组（５％）最高；表现￣（６５）Ｚｎ吸收率，表现Ｚｎ吸收率及表现Ｚｎ潴留率皆随膳食蛋白质水平增加而增加。５％蛋白质水平组明显低于蛋白质≥１０％各组（Ｐ＜０．０１），而１０％、１５％、２０％三组数值相接近，无显著性意义（Ｐ＞０．０５），提示低蛋白降低锌吸收与潴留，增加内源锌排泄；而１０％蛋白质，１５ｍｇ／ｋｇ锌为蛋白质和锌较适宜的膳食含量。在方法学上比较观察了￣（６５）Ｚｎ示踪法和总锌法。结果显示：大鼠肝、肾、胫骨、脑、睾丸各脏器Ｚｎ及￣（６５）Ｚｎ潴留存在明显正相关关系（Ｐ＜０．０５），且表现￣（６５）Ｚｎ吸收率与表现Ｚｎ吸收率、表现Ｚｎ潴留率呈高度正相关Ｐ（＜０．０５），示踪法特异性强，灵敏度高，无内源锌干扰，建议广泛使用，替代总锌法。

测定鸡血细胞和血浆蛋白在体外对锌-65的摄取评定鸡体锌营养状况

本试验探讨鸡血细胞和血浆蛋白在体外对锌-65摄取量作为评定鸡体锌营养状况指标的可行性。试验结果表明,血细胞和血浆蛋白在体外对锌65摄取随饲粮中锌水平提高而下降,二者分别与饲粮中锌水平呈负相关(r=-0.684,-0.315);而锌-65摄取量随饲粮中钙水平提高而增加。与评定鸡体锌营养状况常用指标比较,血细胞在体外对锌-65的摄取作为反映饲粮中含锌状况的指标有较好的灵敏度,且不受血浆中营养成分变化的影响;而血浆蛋白对锌-65摄取灵敏度较差。

肿瘤及胚胎组织特异表达蛋白65与低密度脂蛋白受体相关蛋白关联蛋白的结合

目的:寻找与肿瘤及胚胎组织特异表达蛋白65(cancerandembryoexpressionprotein65,CEP65)相互结合的蛋白分子,为研究CEP65在肿瘤发生中的作用机制提供线索。方法: 以CEP65为诱饵蛋白,通过酵母双杂交方法, 筛选人胚胎组织cDNA表达文库。将获得的阳性克隆cDNA片段分别克隆到原核和哺乳细胞表达质粒,通过GSTpull down和免疫共沉淀对相互作用蛋白作进一步验证。结果: 筛选到能与CEP65相互结合的低密度脂蛋白受体相关蛋白产关联蛋白(lowdensitylipoproteinreceptor relatedprotein associatedprotein1,RAP)。将分别在原核和哺乳细胞中表达的RAP与CEP65进行GSTpull down实验,结果显示,原核表达的GST RAP可以与His CEP65相互结合,而在哺乳细胞共表达的GST CEP65和Myc RAP也能相互结合。结论:RAP可以和CEP65相结合,CEP65有可能通过与RAP相互作用,在肿瘤的浸润、转移及增生中发挥其功能。