重组人硫氧还蛋白1 cDNA的克
发布时间：2018-09-05 16:10
【摘要】：研究背景 内毒素血症(endotoxemia, ETM)是由于体内革兰氏阴性细菌释放出大量内毒素,侵入血液循环而引起的一系列病理生理反应,是引起全身炎症反应综合征(systemic inflammatory response syndrome, SIRS)和多器官功能衰竭(multiple organ failure, MOF)的主要原因。脂多糖(LPS)是内毒素的主要成分,来源于革兰氏阴性菌细胞壁的外膜,由核心寡聚糖、特异性多糖与类脂A组成,其中类脂A是LPS的活性部位以及毒力中心。LPS几乎可以介导所有的内毒素生物学效应,激活机体的免疫应答,促使机体产生多种炎性介质,引发机体的全身炎症反应,进而影响机体屏障功能和脏器功能的完整性,严重时将导致发热反应、白细胞反应以及内毒素血症和内毒素休克等。 严重感染导致的内毒素血症是引起新生儿和儿童全身炎症反应和死亡的主要原因之一,时临床上常见的危重症,其高发病率及死亡率是长期以来困扰医学界的一大难题。在内毒素血症及并发症的治疗方面,目前除了使用抗生素及激素等对症处理外,仍无明显有效的治疗手段。更棘手的食,应用抗生素后,细菌被抑制或者杀死,菌血症虽得到有效控制,但是细菌在杀死的同时,大量的内毒素释放出来,体液或血浆中的内毒素水平升高,机体免疫反应被过度激活,内毒素血症病情会进一步加重。 硫氧还蛋白(thioredoxin, Trx)又称白细胞介素-1样细胞因子、成人T细胞白血病衍化因了和早孕因子,是一种于1964年在大肠杆菌、1989年在人体中分别首次发现的普遍存在于不同有机体的小分子蛋白。它广泛存在于原核生物和真核生物中,其活性位点为-Cys-Gly-Pro-Cys-,通过其特有的二硫化物活性中心,可逆地催化许多氧化还原反应。根据Trx定位,可以将其分为三种：Trx1、Trx2和Trx3。Trxl位于细胞质中,Trx2位于线粒体中,Trx3则主要存在精子细胞的内质网中。 Trx-1是硫氧还蛋白家族中研究最广泛的,是在胞内胞外均存在的多功能蛋白,它除了具有基本的抗氧化功能外,还具有促生长、抗凋亡、抗炎和调节转录因子活性等作用。近年来,国内外研究发现,人硫氧还蛋白1与炎症性疾病关系密切,目前研究较多的是其抗炎的作用以及相关机制。另外,人硫氧还蛋1通过肾脏排泄,除肾脏浓度排第一位外,肺脏的硫氧还蛋白1浓度排在第二位,这提示硫氧还蛋白或许可以成为肺部疾病的重要治疗药物。 目的 制备重组人硫氧还蛋白1(recombinant human thioredoxin-1, rhTrx-1),用此蛋白免疫动物制备抗血清,并对抗血清的效价和特异性进行检测,验证其可用性。通过建立新生大鼠内毒素血症模型并予以rhTrx-1进行干预,初步探讨该重组蛋白是否对内毒素新生大鼠具有保护作用,为后期的实验奠定基础。 方法 1. rhTrx-1cDNA的获取及引物设计： 从人胎肝细胞中提取总RNA,反转录成cDNA。从人的cDNA文库中筛选出hTrx-1基因,根据其编码序列,利用DNA Club软件设计上下游引物。应用设计的特异引物,利用高保真的DNA聚合酶进行扩增,PCR产物利用琼脂糖凝胶电泳鉴定。 2.重组原核表达载体的构建以及鉴定： PCR鉴定回收产物和PET-28a(+)载体分别用SalI和SacI双酶切、回收、连接后转入大肠埃希菌DH5a感受态细胞,卡那霉素筛选阳性克降,提取的重组质粒进行酶切和测序鉴定。 3.重组蛋白的诱导表达： 提取PET-28a(+)-hTrx-1重组质粒转化入大肠埃希菌BL21工程菌,卡那霉素筛选阳性克降,挑取单菌落接种于含卡那霉素的LB液体培养基中,振荡培养过夜并加入IPTG至浓度1mmol/1诱导,离心收集菌体沉淀,处理样品后取上清进行SDS-PAGE电泳分析。 4.重组蛋白的大量诱导和纯化： 根据上述的诱导表达方法确定最适宜的诱导时间,然后对阳性克隆进行大量的诱导表达,离心收集菌体,1×结合Buffer重悬后4℃过夜,次日解冻后于冰上超声裂解后,分别取沉淀和上清样品处理后行SDS-PAGE蛋白电泳判断重组蛋白的可溶性。蛋白在上清表达,去内毒素后参照His柱蛋白纯化说明书进行纯化,目的蛋白透析后,Bradford法进行蛋白浓度测定,并于-80℃保存备用。 5.多克隆抗体的制备与鉴定： 初次免疫时,将200μg蛋白与等体积的弗氏完全佐剂充分混合,于大鼠皮下多点免疫。随后每隔2周,用100pμg蛋白与等体积的弗氏不完全佐剂混合,进行多点加强免疫,同时设立对照组及阴性对照组。每次免疫前及末次免疫两周后大鼠尾部采血,间接ELISA法检测抗血清效价。制备纯化的rhTrx-1多克隆抗体后以Western blot方法检测其可用性以及特异性。 6.对内毒素新生大鼠保护作用的初步观察： 以足月新生7天SD大鼠为实验对象,LPS5mg/kg腹腔内注射,制备新生SD大鼠内毒素血症模型。36只新生7天大鼠随机分为阴性对照组、对照组和实验组,每组各12只,阴性对照组腹腔注射0.9%生理盐水0.1mL；对照组腹腔注射LPS5mg/kg;实验组于LPS注射前30min腹腔注射rhTrx-1(10mg/kg),观察注射后24h各组新生SD大鼠的一般情况以及死亡率。重复实验,每组分别于实验后的2h、8h随机处死数只,提取肺组织用于病理切片观察。 7.采用SPSS13.0统计软件进行分析： 三组间死亡率比较采用多个样本率的χ2检验,P0.05有统计学意义。 结果 1.原核重组质粒的鉴定： 提取原核重组质粒,行PCR扩增和双酶切鉴定,反应后的产物行琼脂糖凝胶电泳。结果显示在600bp左右有一清晰条带,与目的基因大小基本相符。重组质粒的测序报告显示插入序列与理论序列完全一致,证明重组质粒构建成功。 2.最佳诱导时间： 加入IPTG诱导剂后,目的蛋白表达增加,且随着时间的延长,蛋白表达量增多,但4-5h未有明显变化。 3.蛋白表达以及纯化结果： 将构建好的重组质粒转化到大肠埃希菌BL21中,SDS-PAGE电泳显示在大约27.9kDa处出现高效表达条带,与目的蛋白基本相符。经鉴定,蛋白主要存在于上清,经过柱后得到纯化蛋白,其位置与目的蛋白相符,证明目的蛋白纯化成功。 4.间接ELISA法检测多克隆抗体效价： 间接ELISA结果表明,蛋白免疫3次后,第6周时效价达1：51200以上。 5. Western blot法检测抗原抗体特异性结合： 多克降抗体对人硫氧还蛋白1的Western blot结果显示在27.9kDa处呈现单一清晰条带,而正常大鼠血清对该蛋白未识别出条带,证明抗体特异性良好。 6.新生SD大鼠24h死亡率： 阴性对照组活动、饮食一切正常,没有死亡发生：对照组动物注入LPS1h后,表现出活动减少、离群、嗜睡、口周发绀、呼吸急促等全身炎症反应症状,濒死状态时表现为毛色青灰、呼吸浅表,24h死亡率为67%(8/12)；实验组动物症状不明显,24h死亡率为17%(2/12)；3组间死亡率差异有统计学意义(χ2=14.400,P=0.001)。 7.肺组织大体观察： 对照组腹腔注射LPS一段时间后,新生大鼠肺脏可见点状肺出血,随着给药时间延长,出血程度由局灶性到弥漫性逐渐加重。与对照组相比,实验组的新生大鼠肺脏出血程度明显减轻。 8.肺组织病理形态学观察： 阴性对照组肺组织结构完整,肺泡隔无水肿,肺泡腔清晰。对照组肺组织结构不完整,肺泡腔内有大量红细胞、炎性细胞和浆液渗出,血管周围组织可见白细胞浸润,毛细血管有明显充血及扩张表现,肺泡间隔明显增宽。实验组病理改变较对照组明显减轻,肺泡结构尚可,轻度肺水肿,肺泡间隔稍增宽,红细胞渗出明显减少,肺泡及肺间质中白细胞浸润减少。 结论 1.本研究中,首先合成了人硫氧还蛋白1的原核重组质粒,通过PCR、双酶切以及测序鉴定,均证明重组质粒构建成功。接着将重组质粒转化到大肠杆菌BL21,进行人硫氧还蛋白的原核表达,对表达出的蛋白进行SDS-PAGE鉴定,其分子量大小与通过碱基数计算出来的大小一致,提示人硫氧还蛋白1的原核表达成功。 2.用此蛋白免疫SD大鼠制备的抗血清,经Elisa检测,效价达到1：51200以上,进一步用Western blot检测,证明特异性良好,所制备的多克隆抗体能与人硫氧还蛋白1特异性结合。 3.用此重组蛋白干预内毒素血症新生SD大鼠,动物的24小时死亡率明显降低,肺出血明显减轻,肺组织病理形态观察提示红细胞和炎症细胞的渗出及浸润减少,这些初步提示rhTrx-1对内毒素血症新生大鼠具有保护作用,为后期的实验奠定了基础。
[Abstract]:Research background
Endotoxemia (ETM) is a series of pathophysiological reactions caused by the release of large quantities of endotoxin from Gram-negative bacteria and the invasion of blood circulation. It is the main cause of systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Lipopolysaccharide (LPS) is the main component of endotoxin. It originates from the outer membrane of the cell wall of Gram-negative bacteria and consists of core oligosaccharides, specific polysaccharides and lipoid A. Lipopolysaccharide A is the active site and toxicity center of LPS. LPS can mediate almost all the biological effects of endotoxin, activate the body's immune response and promote the production of the body. It produces a variety of inflammatory mediators, triggering systemic inflammation, thereby affecting the integrity of the body's barrier function and organ function. In severe cases, it will lead to fever, leukocyte reaction, endotoxemia and endotoxin shock.
Endotoxemia caused by severe infections is one of the main causes of systemic inflammation and death in newborns and children. It is a common clinical critical illness. The high incidence and mortality of endotoxemia have long been a major problem in the medical community. In the treatment of endotoxemia and complications, antibiotics and hormones are currently used in addition to antibiotics and hormones. The more difficult food, after the application of antibiotics, bacteria are inhibited or killed, although bacteremia has been effectively controlled, but bacteria kill at the same time, a large number of endotoxins released, body fluid or plasma endotoxin levels increased, the body's immune response was over-activated, endotoxin blood. The disease will further aggravate.
Thioredoxin (Trx), also known as interleukin-1-like cytokines, is an adult T-cell leukemia-derived and early pregnancy factor. It is a small molecule protein first found in E. coli in 1964 and in human body in 1989. It is widely found in prokaryotes and eukaryotes. The active site, Cys-Gly-Pro-Cys-, catalyzes many redox reactions reversibly through its unique disulfide active site. According to Trx localization, it can be divided into three types: Trx1, Trx2 and Trx3. Trxl are located in the cytoplasm, Trx2 in the mitochondria, and Trx3 in the endoplasmic reticulum of sperm cells.
Trx-1 is one of the most extensively studied thioredoxins, which is a multifunctional protein both inside and outside the cell. Besides its basic antioxidant function, it also has the functions of promoting growth, anti-apoptosis, anti-inflammatory and regulating transcription factors. In addition, human thioredoxin-1 is excreted through the kidney, and the lung thioredoxin-1 is in the second place besides the kidney, suggesting that thioredoxin may be an important therapeutic drug for lung diseases.
objective
Recombinant human thioredoxin-1 (rhTrx-1) was prepared and used to immunize animals to prepare antiserum. The antiserum titer and specificity were tested to verify its feasibility. The endotoxemia model of neonatal rats was established and rhTrx-1 was intervened to explore whether the recombinant protein was endotoxic. The newborn rats have protective effect and lay the foundation for later experiments.
Method
1. rhTrx-1cDNA acquisition and primer design:
Total RNA was extracted from human fetal liver cells and transcribed into cDNA. The hTrx-1 gene was screened out from the human cDNA library. According to its coding sequence, the upstream and downstream primers were designed by DNA Club software. The designed specific primers were used to amplify by high-fidelity DNA polymerase, and the PCR products were identified by agarose gel electrophoresis.
2. construction and identification of Recombinant Prokaryotic expression vector:
Recovered products and PET-28a (+) vectors were digested with SalI and Saci, recovered, linked and transfected into E.coli DH5a competent cells. Kanamycin was screened positive, and the recombinant plasmids were identified by enzyme digestion and sequencing.
3. expression of recombinant protein:
The recombinant plasmid PET-28a(+) -hTrx-1 was extracted and transformed into Escherichia coli BL21 strain. Kanamycin was screened for positive control. The single colony was inoculated in the liquid medium containing kanamycin. After shaking culture overnight, IPTG was added into the medium to induce 1 mmol/1 concentration, the bacteria were centrifuged to collect precipitation and the supernatant was taken for SDS-PAGE analysis.
4. large amount of induction and purification of recombinant protein:
The optimum induction time was determined according to the above methods. The positive clones were induced to express in large quantities. The bacteria were centrifuged and collected. The cells were suspended at 4 C after 1 combined with Buffer. After thawing the next day, they were lysed by ultrasound on ice. After precipitation and supernatant were treated, the solubility of the recombinant protein was determined by SDS-PAGE electrophoresis. The protein was expressed in supernatant and purified according to His column protein purification instructions after removing endotoxin. After dialysis, the protein concentration was determined by Bradford method and stored at - 80 C for reserve.
5. preparation and identification of polyclonal antibodies:
At the first immunization, 200 UG protein was fully mixed with the same volume of Freund's complete adjuvant and immunized subcutaneously at multiple sites in rats. The purified rhTrx-1 polyclonal antibody was prepared and tested by Western blot.
Protective effects of 6. on endotoxin in neonatal rats:
The endotoxemia model of neonatal SD rats was established by intraperitoneal injection of LPS 5 mg/kg in full-term neonatal rats of 7 days. 36 neonatal SD rats of 7 days were randomly divided into negative control group, control group and experimental group, 12 rats in each group, 0.9% normal saline 0.1 mL was injected intraperitoneally in negative control group, LPS 5 mg/kg was injected intraperitoneally in control group, and LPS 5 mg/kg was injected into experimental group. RhTrx-1 (10mg/kg) was injected intraperitoneally in the first 30 minutes to observe the general condition and mortality of neonatal SD rats 24 hours after injection.
7. SPSS13.0 statistical software was used to analyze:
The mortality rates between the three groups were compared by chi square test with multiple sample rates, and the P0.05 was statistically significant.
Result
1. identification of prokaryotic recombinant plasmids:
The Prokaryotic Recombinant plasmid was extracted, amplified by PCR and identified by double enzyme digestion, and the product was identified by agarose gel electrophoresis. The results showed that there was a clear band about 600 bp, which was basically consistent with the size of the target gene.
2. the best induction time:
After adding IPTG inducer, the expression of the target protein increased, and with the prolongation of time, the expression of the target protein increased, but there was no significant change in 4-5 H.
3. protein expression and purification results:
The recombinant plasmid was transformed into Escherichia coli BL21. SDS-PAGE electrophoresis showed that a high expression band appeared at about 27.9 kDa, which was consistent with the target protein.
4. indirect ELISA method was used to detect the titer of polyclonal antibody.
Indirect ELISA results showed that after 3 times of protein immunization, the aging rate was more than 1:51200 at sixth weeks.
5. Western blot method was used to detect the specific binding of antigen and antibody.
The Western blot results of Dog antibody against human thioredoxin 1 showed a single clear band at 27.9 kDa, but the normal rat serum did not recognize the band, which proved that the specificity of the antibody was good.
6. 24h mortality of neonatal SD rats:
Negative control group was active, diet was normal, no death occurred: control group animals injected LPS 1 hour, showed reduced activity, isolation, drowsiness, perioral cyanosis, shortness of breath and other systemic inflammatory reaction symptoms, near-death state of hair grey, shallow breathing, 24-hour mortality rate was 67% (8/12); experimental group animal symptoms were not obvious, 24-hour death. The rate was 17% (2/12), and the difference between the 3 groups was statistically significant (x 2=14.400, P=0.001).
7. gross observation of lung tissue:
In the control group, punctate pulmonary hemorrhage was observed in the lungs of neonatal rats after intraperitoneal injection of LPS for a period of time.
8. pathological observation of lung tissue:
In the negative control group, the pulmonary tissue was intact, the alveolar septum was edema free, and the alveolar cavity was clear. In the control group, the alveolar structure was still normal, mild pulmonary edema, alveolar septum widened slightly, erythrocyte exudation decreased significantly, and leukocyte infiltration in alveoli and interstitium decreased.
conclusion
1. In this study, the recombinant plasmid of human thioredoxin 1 was synthesized, and the recombinant plasmid was successfully constructed by PCR, double enzyme digestion and sequencing. Then the recombinant plasmid was transformed into E. coli BL21 for prokaryotic expression of human thioredoxin, and the expressed protein was identified by SDS-PAGE. The same size of the base number indicates that the prokaryotic expression of human thioredoxin 1 is successful.
2. The antiserum prepared from SD rats was immunized with this protein and its titer was over 1:51200 by Elisa. The specificity of the antiserum was confirmed by Western blot. The polyclonal antibody could bind to human thioredoxin 1.
3. The 24-hour mortality and pulmonary hemorrhage of neonatal SD rats with endotoxemia were significantly reduced, and the infiltration and exudation of erythrocytes and inflammatory cells were decreased. These preliminary results suggested that rhTrx-1 had protective effect on neonatal rats with endotoxemia and laid a foundation for the later experiment. The foundation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1
本文编号：2224782
[Abstract]:Research background
Endotoxemia (ETM) is a series of pathophysiological reactions caused by the release of large quantities of endotoxin from Gram-negative bacteria and the invasion of blood circulation. It is the main cause of systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Lipopolysaccharide (LPS) is the main component of endotoxin. It originates from the outer membrane of the cell wall of Gram-negative bacteria and consists of core oligosaccharides, specific polysaccharides and lipoid A. Lipopolysaccharide A is the active site and toxicity center of LPS. LPS can mediate almost all the biological effects of endotoxin, activate the body's immune response and promote the production of the body. It produces a variety of inflammatory mediators, triggering systemic inflammation, thereby affecting the integrity of the body's barrier function and organ function. In severe cases, it will lead to fever, leukocyte reaction, endotoxemia and endotoxin shock.
Endotoxemia caused by severe infections is one of the main causes of systemic inflammation and death in newborns and children. It is a common clinical critical illness. The high incidence and mortality of endotoxemia have long been a major problem in the medical community. In the treatment of endotoxemia and complications, antibiotics and hormones are currently used in addition to antibiotics and hormones. The more difficult food, after the application of antibiotics, bacteria are inhibited or killed, although bacteremia has been effectively controlled, but bacteria kill at the same time, a large number of endotoxins released, body fluid or plasma endotoxin levels increased, the body's immune response was over-activated, endotoxin blood. The disease will further aggravate.
Thioredoxin (Trx), also known as interleukin-1-like cytokines, is an adult T-cell leukemia-derived and early pregnancy factor. It is a small molecule protein first found in E. coli in 1964 and in human body in 1989. It is widely found in prokaryotes and eukaryotes. The active site, Cys-Gly-Pro-Cys-, catalyzes many redox reactions reversibly through its unique disulfide active site. According to Trx localization, it can be divided into three types: Trx1, Trx2 and Trx3. Trxl are located in the cytoplasm, Trx2 in the mitochondria, and Trx3 in the endoplasmic reticulum of sperm cells.
Trx-1 is one of the most extensively studied thioredoxins, which is a multifunctional protein both inside and outside the cell. Besides its basic antioxidant function, it also has the functions of promoting growth, anti-apoptosis, anti-inflammatory and regulating transcription factors. In addition, human thioredoxin-1 is excreted through the kidney, and the lung thioredoxin-1 is in the second place besides the kidney, suggesting that thioredoxin may be an important therapeutic drug for lung diseases.
objective
Recombinant human thioredoxin-1 (rhTrx-1) was prepared and used to immunize animals to prepare antiserum. The antiserum titer and specificity were tested to verify its feasibility. The endotoxemia model of neonatal rats was established and rhTrx-1 was intervened to explore whether the recombinant protein was endotoxic. The newborn rats have protective effect and lay the foundation for later experiments.
Method
1. rhTrx-1cDNA acquisition and primer design:
Total RNA was extracted from human fetal liver cells and transcribed into cDNA. The hTrx-1 gene was screened out from the human cDNA library. According to its coding sequence, the upstream and downstream primers were designed by DNA Club software. The designed specific primers were used to amplify by high-fidelity DNA polymerase, and the PCR products were identified by agarose gel electrophoresis.
2. construction and identification of Recombinant Prokaryotic expression vector:
Recovered products and PET-28a (+) vectors were digested with SalI and Saci, recovered, linked and transfected into E.coli DH5a competent cells. Kanamycin was screened positive, and the recombinant plasmids were identified by enzyme digestion and sequencing.
3. expression of recombinant protein:
The recombinant plasmid PET-28a(+) -hTrx-1 was extracted and transformed into Escherichia coli BL21 strain. Kanamycin was screened for positive control. The single colony was inoculated in the liquid medium containing kanamycin. After shaking culture overnight, IPTG was added into the medium to induce 1 mmol/1 concentration, the bacteria were centrifuged to collect precipitation and the supernatant was taken for SDS-PAGE analysis.
4. large amount of induction and purification of recombinant protein:
The optimum induction time was determined according to the above methods. The positive clones were induced to express in large quantities. The bacteria were centrifuged and collected. The cells were suspended at 4 C after 1 combined with Buffer. After thawing the next day, they were lysed by ultrasound on ice. After precipitation and supernatant were treated, the solubility of the recombinant protein was determined by SDS-PAGE electrophoresis. The protein was expressed in supernatant and purified according to His column protein purification instructions after removing endotoxin. After dialysis, the protein concentration was determined by Bradford method and stored at - 80 C for reserve.
5. preparation and identification of polyclonal antibodies:
At the first immunization, 200 UG protein was fully mixed with the same volume of Freund's complete adjuvant and immunized subcutaneously at multiple sites in rats. The purified rhTrx-1 polyclonal antibody was prepared and tested by Western blot.
Protective effects of 6. on endotoxin in neonatal rats:
The endotoxemia model of neonatal SD rats was established by intraperitoneal injection of LPS 5 mg/kg in full-term neonatal rats of 7 days. 36 neonatal SD rats of 7 days were randomly divided into negative control group, control group and experimental group, 12 rats in each group, 0.9% normal saline 0.1 mL was injected intraperitoneally in negative control group, LPS 5 mg/kg was injected intraperitoneally in control group, and LPS 5 mg/kg was injected into experimental group. RhTrx-1 (10mg/kg) was injected intraperitoneally in the first 30 minutes to observe the general condition and mortality of neonatal SD rats 24 hours after injection.
7. SPSS13.0 statistical software was used to analyze:
The mortality rates between the three groups were compared by chi square test with multiple sample rates, and the P0.05 was statistically significant.
Result
1. identification of prokaryotic recombinant plasmids:
The Prokaryotic Recombinant plasmid was extracted, amplified by PCR and identified by double enzyme digestion, and the product was identified by agarose gel electrophoresis. The results showed that there was a clear band about 600 bp, which was basically consistent with the size of the target gene.
2. the best induction time:
After adding IPTG inducer, the expression of the target protein increased, and with the prolongation of time, the expression of the target protein increased, but there was no significant change in 4-5 H.
3. protein expression and purification results:
The recombinant plasmid was transformed into Escherichia coli BL21. SDS-PAGE electrophoresis showed that a high expression band appeared at about 27.9 kDa, which was consistent with the target protein.
4. indirect ELISA method was used to detect the titer of polyclonal antibody.
Indirect ELISA results showed that after 3 times of protein immunization, the aging rate was more than 1:51200 at sixth weeks.
5. Western blot method was used to detect the specific binding of antigen and antibody.
The Western blot results of Dog antibody against human thioredoxin 1 showed a single clear band at 27.9 kDa, but the normal rat serum did not recognize the band, which proved that the specificity of the antibody was good.
6. 24h mortality of neonatal SD rats:
Negative control group was active, diet was normal, no death occurred: control group animals injected LPS 1 hour, showed reduced activity, isolation, drowsiness, perioral cyanosis, shortness of breath and other systemic inflammatory reaction symptoms, near-death state of hair grey, shallow breathing, 24-hour mortality rate was 67% (8/12); experimental group animal symptoms were not obvious, 24-hour death. The rate was 17% (2/12), and the difference between the 3 groups was statistically significant (x 2=14.400, P=0.001).
7. gross observation of lung tissue:
In the control group, punctate pulmonary hemorrhage was observed in the lungs of neonatal rats after intraperitoneal injection of LPS for a period of time.
8. pathological observation of lung tissue:
In the negative control group, the pulmonary tissue was intact, the alveolar septum was edema free, and the alveolar cavity was clear. In the control group, the alveolar structure was still normal, mild pulmonary edema, alveolar septum widened slightly, erythrocyte exudation decreased significantly, and leukocyte infiltration in alveoli and interstitium decreased.
conclusion
1. In this study, the recombinant plasmid of human thioredoxin 1 was synthesized, and the recombinant plasmid was successfully constructed by PCR, double enzyme digestion and sequencing. Then the recombinant plasmid was transformed into E. coli BL21 for prokaryotic expression of human thioredoxin, and the expressed protein was identified by SDS-PAGE. The same size of the base number indicates that the prokaryotic expression of human thioredoxin 1 is successful.
2. The antiserum prepared from SD rats was immunized with this protein and its titer was over 1:51200 by Elisa. The specificity of the antiserum was confirmed by Western blot. The polyclonal antibody could bind to human thioredoxin 1.
3. The 24-hour mortality and pulmonary hemorrhage of neonatal SD rats with endotoxemia were significantly reduced, and the infiltration and exudation of erythrocytes and inflammatory cells were decreased. These preliminary results suggested that rhTrx-1 had protective effect on neonatal rats with endotoxemia and laid a foundation for the later experiment. The foundation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1
【参考文献】
相关期刊论文 前2条
1 蔡栩栩,刘春峰,杜悦,韩晓华,尚云晓,韩玉昆;内毒素致新生和成年大鼠急性肺损伤的比较[J];中华急诊医学杂志;2005年06期
2 张永一;郭昌星;;抗生素诱导内毒素血症研究进展[J];中华全科医学;2008年12期
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