重組人硫氧還蛋白1 cDNA的克
發(fā)布時間：2018-09-05 16:10
【摘要】：研究背景 內毒素血癥(endotoxemia, ETM)是由于體內革蘭氏陰性細菌釋放出大量內毒素,侵入血液循環(huán)而引起的一系列病理生理反應,是引起全身炎癥反應綜合征(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)是內毒素的主要成分,來源于革蘭氏陰性菌細胞壁的外膜,由核心寡聚糖、特異性多糖與類脂A組成,其中類脂A是LPS的活性部位以及毒力中心。LPS幾乎可以介導所有的內毒素生物學效應,激活機體的免疫應答,促使機體產生多種炎性介質,引發(fā)機體的全身炎癥反應,進而影響機體屏障功能和臟器功能的完整性,嚴重時將導致發(fā)熱反應、白細胞反應以及內毒素血癥和內毒素休克等。 嚴重感染導致的內毒素血癥是引起新生兒和兒童全身炎癥反應和死亡的主要原因之一,時臨床上常見的危重癥,其高發(fā)病率及死亡率是長期以來困擾醫(yī)學界的一大難題。在內毒素血癥及并發(fā)癥的治療方面,目前除了使用抗生素及激素等對癥處理外,仍無明顯有效的治療手段。更棘手的食,應用抗生素后,細菌被抑制或者殺死,菌血癥雖得到有效控制,但是細菌在殺死的同時,大量的內毒素釋放出來,體液或血漿中的內毒素水平升高,機體免疫反應被過度激活,內毒素血癥病情會進一步加重。 硫氧還蛋白(thioredoxin, Trx)又稱白細胞介素-1樣細胞因子、成人T細胞白血病衍化因了和早孕因子,是一種于1964年在大腸桿菌、1989年在人體中分別首次發(fā)現(xiàn)的普遍存在于不同有機體的小分子蛋白。它廣泛存在于原核生物和真核生物中,其活性位點為-Cys-Gly-Pro-Cys-,通過其特有的二硫化物活性中心,可逆地催化許多氧化還原反應。根據(jù)Trx定位,可以將其分為三種：Trx1、Trx2和Trx3。Trxl位于細胞質中,Trx2位于線粒體中,Trx3則主要存在精子細胞的內質網中。 Trx-1是硫氧還蛋白家族中研究最廣泛的,是在胞內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,還具有促生長、抗凋亡、抗炎和調節(jié)轉錄因子活性等作用。近年來,國內外研究發(fā)現(xiàn),人硫氧還蛋白1與炎癥性疾病關系密切,目前研究較多的是其抗炎的作用以及相關機制。另外,人硫氧還蛋1通過腎臟排泄,除腎臟濃度排第一位外,肺臟的硫氧還蛋白1濃度排在第二位,這提示硫氧還蛋白或許可以成為肺部疾病的重要治療藥物。 目的 制備重組人硫氧還蛋白1(recombinant human thioredoxin-1, rhTrx-1),用此蛋白免疫動物制備抗血清,并對抗血清的效價和特異性進行檢測,驗證其可用性。通過建立新生大鼠內毒素血癥模型并予以rhTrx-1進行干預,初步探討該重組蛋白是否對內毒素新生大鼠具有保護作用,為后期的實驗奠定基礎。 方法 1. rhTrx-1cDNA的獲取及引物設計： 從人胎肝細胞中提取總RNA,反轉錄成cDNA。從人的cDNA文庫中篩選出hTrx-1基因,根據(jù)其編碼序列,利用DNA Club軟件設計上下游引物。應用設計的特異引物,利用高保真的DNA聚合酶進行擴增,PCR產物利用瓊脂糖凝膠電泳鑒定。 2.重組原核表達載體的構建以及鑒定： PCR鑒定回收產物和PET-28a(+)載體分別用SalI和SacI雙酶切、回收、連接后轉入大腸埃希菌DH5a感受態(tài)細胞,卡那霉素篩選陽性克降,提取的重組質粒進行酶切和測序鑒定。 3.重組蛋白的誘導表達： 提取PET-28a(+)-hTrx-1重組質粒轉化入大腸埃希菌BL21工程菌,卡那霉素篩選陽性克降,挑取單菌落接種于含卡那霉素的LB液體培養(yǎng)基中,振蕩培養(yǎng)過夜并加入IPTG至濃度1mmol/1誘導,離心收集菌體沉淀,處理樣品后取上清進行SDS-PAGE電泳分析。 4.重組蛋白的大量誘導和純化： 根據(jù)上述的誘導表達方法確定最適宜的誘導時間,然后對陽性克隆進行大量的誘導表達,離心收集菌體,1×結合Buffer重懸后4℃過夜,次日解凍后于冰上超聲裂解后,分別取沉淀和上清樣品處理后行SDS-PAGE蛋白電泳判斷重組蛋白的可溶性。蛋白在上清表達,去內毒素后參照His柱蛋白純化說明書進行純化,目的蛋白透析后,Bradford法進行蛋白濃度測定,并于-80℃保存?zhèn)溆谩?5.多克隆抗體的制備與鑒定： 初次免疫時,將200μg蛋白與等體積的弗氏完全佐劑充分混合,于大鼠皮下多點免疫。隨后每隔2周,用100pμg蛋白與等體積的弗氏不完全佐劑混合,進行多點加強免疫,同時設立對照組及陰性對照組。每次免疫前及末次免疫兩周后大鼠尾部采血,間接ELISA法檢測抗血清效價。制備純化的rhTrx-1多克隆抗體后以Western blot方法檢測其可用性以及特異性。 6.對內毒素新生大鼠保護作用的初步觀察： 以足月新生7天SD大鼠為實驗對象,LPS5mg/kg腹腔內注射,制備新生SD大鼠內毒素血癥模型。36只新生7天大鼠隨機分為陰性對照組、對照組和實驗組,每組各12只,陰性對照組腹腔注射0.9%生理鹽水0.1mL；對照組腹腔注射LPS5mg/kg;實驗組于LPS注射前30min腹腔注射rhTrx-1(10mg/kg),觀察注射后24h各組新生SD大鼠的一般情況以及死亡率。重復實驗,每組分別于實驗后的2h、8h隨機處死數(shù)只,提取肺組織用于病理切片觀察。 7.采用SPSS13.0統(tǒng)計軟件進行分析： 三組間死亡率比較采用多個樣本率的χ2檢驗,P0.05有統(tǒng)計學意義。 結果 1.原核重組質粒的鑒定： 提取原核重組質粒,行PCR擴增和雙酶切鑒定,反應后的產物行瓊脂糖凝膠電泳。結果顯示在600bp左右有一清晰條帶,與目的基因大小基本相符。重組質粒的測序報告顯示插入序列與理論序列完全一致,證明重組質粒構建成功。 2.最佳誘導時間： 加入IPTG誘導劑后,目的蛋白表達增加,且隨著時間的延長,蛋白表達量增多,但4-5h未有明顯變化。 3.蛋白表達以及純化結果： 將構建好的重組質粒轉化到大腸埃希菌BL21中,SDS-PAGE電泳顯示在大約27.9kDa處出現(xiàn)高效表達條帶,與目的蛋白基本相符。經鑒定,蛋白主要存在于上清,經過柱后得到純化蛋白,其位置與目的蛋白相符,證明目的蛋白純化成功。 4.間接ELISA法檢測多克隆抗體效價： 間接ELISA結果表明,蛋白免疫3次后,第6周時效價達1：51200以上。 5. Western blot法檢測抗原抗體特異性結合： 多克降抗體對人硫氧還蛋白1的Western blot結果顯示在27.9kDa處呈現(xiàn)單一清晰條帶,而正常大鼠血清對該蛋白未識別出條帶,證明抗體特異性良好。 6.新生SD大鼠24h死亡率： 陰性對照組活動、飲食一切正常,沒有死亡發(fā)生：對照組動物注入LPS1h后,表現(xiàn)出活動減少、離群、嗜睡、口周發(fā)紺、呼吸急促等全身炎癥反應癥狀,瀕死狀態(tài)時表現(xiàn)為毛色青灰、呼吸淺表,24h死亡率為67%(8/12)；實驗組動物癥狀不明顯,24h死亡率為17%(2/12)；3組間死亡率差異有統(tǒng)計學意義(χ2=14.400,P=0.001)。 7.肺組織大體觀察： 對照組腹腔注射LPS一段時間后,新生大鼠肺臟可見點狀肺出血,隨著給藥時間延長,出血程度由局灶性到彌漫性逐漸加重。與對照組相比,實驗組的新生大鼠肺臟出血程度明顯減輕。 8.肺組織病理形態(tài)學觀察： 陰性對照組肺組織結構完整,肺泡隔無水腫,肺泡腔清晰。對照組肺組織結構不完整,肺泡腔內有大量紅細胞、炎性細胞和漿液滲出,血管周圍組織可見白細胞浸潤,毛細血管有明顯充血及擴張表現(xiàn),肺泡間隔明顯增寬。實驗組病理改變較對照組明顯減輕,肺泡結構尚可,輕度肺水腫,肺泡間隔稍增寬,紅細胞滲出明顯減少,肺泡及肺間質中白細胞浸潤減少。 結論 1.本研究中,首先合成了人硫氧還蛋白1的原核重組質粒,通過PCR、雙酶切以及測序鑒定,均證明重組質粒構建成功。接著將重組質粒轉化到大腸桿菌BL21,進行人硫氧還蛋白的原核表達,對表達出的蛋白進行SDS-PAGE鑒定,其分子量大小與通過堿基數(shù)計算出來的大小一致,提示人硫氧還蛋白1的原核表達成功。 2.用此蛋白免疫SD大鼠制備的抗血清,經Elisa檢測,效價達到1：51200以上,進一步用Western blot檢測,證明特異性良好,所制備的多克隆抗體能與人硫氧還蛋白1特異性結合。 3.用此重組蛋白干預內毒素血癥新生SD大鼠,動物的24小時死亡率明顯降低,肺出血明顯減輕,肺組織病理形態(tài)觀察提示紅細胞和炎癥細胞的滲出及浸潤減少,這些初步提示rhTrx-1對內毒素血癥新生大鼠具有保護作用,為后期的實驗奠定了基礎。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.1
本文編號：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.1
【參考文獻】
相關期刊論文 前2條
1 蔡栩栩,劉春峰,杜悅,韓曉華,尚云曉,韓玉昆;內毒素致新生和成年大鼠急性肺損傷的比較[J];中華急診醫(yī)學雜志;2005年06期
2 張永一;郭昌星;;抗生素誘導內毒素血癥研究進展[J];中華全科醫(yī)學;2008年12期
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