本文選題：Neurocan基因 + 原核表達　； 參考：《南方醫科大學》2009年碩士論文

【摘要】： 中樞神經系統(Central Nervous System,CNS)損傷后的修復受多種因素影響,其中抑制因子釋放和膠質瘢痕形成為主要原因之一。目前已經分離和鑒定的軸突生長抑制因子有:Nogo-A、髓磷脂相關糖蛋白(myelin-associatedglycoprotein,MAG)、少突膠質細胞髓磷脂糖蛋白(oligodendrocyte-myelinglycoprotein,OMgp)等。研究發現,Nogo-A、MAG、OMgp發生作用時都有LINGO-1蛋白的參與。在膠質瘢痕對軸突再生的影響中,硫酸軟骨素蛋白多糖(Chondroitin Sulphate Proteoglycans,GSPGs)是目前已知的一個阻礙軸突再生的重要因子,特異存在于CNS,而Neurocan則為GSPGs的核心蛋白組分之一,并且顯示了對視網膜神經節細胞神經突生長的抑制作用。腱糖蛋白-R(tenascin-R,TN-R)是瘢痕組織中所含有的另一種有軸突生長抑制作用的細胞外基質成分。把LINGO-1、Neurocan和TN-R設計成三基因聯合DNA疫苗,將會使疫苗在體內發揮作用后、產生抗體并與相關抑制因子結合,以消除抑制作用、促進神經再生。本研究則主要是進行Neurocan基因的蛋白表達及其抗體制備,并且對其進行檢測,探討DNA疫苗制作前期技術方案的可行性,為后續CNS損傷修復實驗工作奠定基礎。 目的 制備Neurocan蛋白,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,最終使待參與DNA疫苗構成的Neurocan蛋白所產生抗體能與腦內創傷區的相應抑制性蛋白抗原結合。 方法 從基因庫調出Neurocan的基因序列,合成Neurocan基因,序列起始端加His標簽,兩端設計酶切位點。構建原核表達質粒PET30a(+)-Neurocan,對合成的Neurocan基因進行測序鑒定。將質粒轉化到大腸桿菌BL21感受態,挑菌培養,使用OMEGA公司質粒提取試劑盒(E.Z.N.A.~(TM)Plasmid Mini KitⅡ50)提取質粒。對質粒進行酶切鑒定和PCR鑒定,鑒定正確后,對菌株進行異丙基-β-D-硫代半乳糖苷(Isopropylβ-D-1- Thiogalactopyranoside,IPTG)誘導表達,采用不同的誘導時間和IPTG濃度,確定最佳表達條件。對目的蛋白進行純化,使用PIERCE公司的BCA~(TM) Protein Assay Kit試劑盒測定蛋白濃度。對目的蛋白進行SDS-PAGE檢測,觀察目的蛋白的純度和分子量大小;對目的蛋白進行Western blot鑒定,一抗使用小鼠抗His抗體,二抗使用馬抗小鼠—AP抗體,顯色底物為NBT/BCIP。Neurocan蛋白免疫兔制備免疫血清,分4次免疫,皮內注射。Elisa法檢測抗血清效價,以Neurocan蛋白包被酶標板,兔免疫前血清為陰性對照,二抗為山羊抗兔-HRP,底物顯色后酶標儀檢測;Western blot檢測抗血清特異性,以免疫血清為一抗,山羊抗兔-AP為二抗,NBT/BCIP顯色。 結果 合成的Neurocan基因經測序鑒定,顯示序列正確,且開放讀碼框正確。Neurocan基因經連接、轉化、誘導表達后,測得蛋白濃度為0.673ug%qul。目的蛋白經SDS-PAGE鑒定,目的條帶分子量大小約為55kD;經Western blot鑒定,在55kD處有特異性條帶,與SDS-PAGE鑒定結果一致,目的蛋白能與His抗體特異性結合。制備的抗血清經Elisa法檢測,效價達到1:100萬;經Westernblot檢測,在55kD處的目的條帶明顯。 討論 本研究中,首先對合成的Neurocan基因進行了測序鑒定,顯示其序列正確,且開放讀碼框和酶切位點與本課題設計的一致。接著將包含Neurocan基因的質粒PET30a(+)-Neurocan轉化到大腸桿菌BL21,進行Neurocan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計算出來的大小一致。進一步對其進行Western blot鑒定,因為本實驗在Neurocan基因的起始端設計了His標簽,故一抗用His抗體,結果顯示目的蛋白能與His抗體特異性結合,說明該目的蛋白是Neurocan基因的表達產物,而Neurocan基因經過測序鑒定正確,提示Neurocan蛋白的原核表達成功。用此蛋白免疫兔制備的抗血清,經Elisa檢測,效價達到1:100萬;進一步用Western blot檢測,都證明特異性良好,所制備的多克隆抗體能與Neurocan蛋白特異性結合。因此提示,Neurocan蛋白原核表達成功;用Neurocan蛋白免疫兔,產生的抗體能與Neurocan蛋白特異性結合,為DNA疫苗的進一步研制及CNS損傷后修復策略提供了有意義的參考依據。
[Abstract]:The repair of Central Nervous System (CNS) after injury is affected by a variety of factors, in which inhibition factor release and glial scar form become one of the main reasons. There are currently isolated and identified axon growth inhibitors: Nogo-A, myelin related glycoprotein (myelin-associatedglycoprotein, MAG), oligodendrocyte pulp Oligodendrocyte-myelinglycoprotein (OMgp) and so on. Studies have found that Nogo-A, MAG, and OMgp are involved in the participation of LINGO-1 protein. In the effect of glial scar on axon regeneration, the chondroitin sulfate proteoglycan (Chondroitin Sulphate Proteoglycans, GSPGs) is an important factor that is known to impede axonal regeneration. It is specific to CNS, and Neurocan is one of the core protein components of GSPGs and shows the inhibitory effect on the growth of the retinal ganglion cells. -R (tenascin-R, TN-R) is another extracellular matrix component of the inhibition of axon growth in scar tissue. LINGO-1, Neurocan and TN-R are set up. The combination of the three gene DNA vaccine will enable the vaccine to play a role in the body, produce antibodies and combine with related inhibitory factors to eliminate inhibition and promote nerve regeneration. This study is mainly about the protein expression and antibody preparation of the Neurocan gene, and the detection of it and the preliminary technical scheme for the preparation of the DNA vaccine. The feasibility lays the foundation for subsequent CNS damage repair experiments.
objective
The Neurocan protein was prepared, the antiserum was prepared by this protein, and the titer and specificity of the serum were detected. Finally, the antibody produced by the Neurocan protein, which is made up of the DNA vaccine, could be combined with the corresponding inhibitory protein antigen in the brain wound area.
Method
The gene sequence of Neurocan was transferred from the gene pool, the Neurocan gene was synthesized, the initial end of the sequence was added with the His label, the enzyme cutting site was designed at both ends. The prokaryotic expression plasmid PET30a (+) -Neurocan was constructed, and the synthesized Neurocan gene was sequenced and identified. The plasmid was transformed into the BL21 sense of Escherichia coli, picked up the bacteria and used the OMEGA company plasmid to extract the kit. The plasmid was extracted (E.Z.N.A.~ (TM) Plasmid Mini Kit II 50). The plasmid was identified by enzyme digestion and PCR identification. After the identification was correct, the strain was induced by isopropyl - beta -D- Thioglucoside (Isopropyl beta -D-1- Thiogalactopyranoside, IPTG). The optimal expression conditions were determined by different inducible time and IPTG concentration. Purified, the protein concentration was measured by PIERCE's BCA~ (TM) Protein Assay Kit kit. The pureness and molecular weight of the target protein were detected by SDS-PAGE and the purity and molecular weight of the target protein were observed. The target protein was identified with Western blot, the first anti His antibody was used, the two anti horse anti mouse mouse AP antibody was used, and the chromogenic substrate was NBT/BCIP.Neur. Ocan protein immunized rabbits were immunized to prepare immunized serum, 4 times of immunization, intradermal injection of.Elisa to detect antiserum titer, Neurocan protein was coated with enzyme labeled plate, rabbit pre immunized serum was negative control, two anti Rabbit Rabbit anti rabbit -HRP, the substrate chromogenic enzyme labeling instrument was detected, Western blot was used to detect anti serum specificity, immune serum was one anti, Goat anti rabbit -AP was Two anti, NBT/BCIP color.
Result
The synthesized Neurocan gene was sequenced and identified, and the sequence was correct, and the correct.Neurocan gene of the open reading frame was connected, transformed and induced, and the protein concentration was identified as 0.673ug%qul. by SDS-PAGE. The molecular weight of the target band was about 55kD; the specific bands at 55kD were identified by Western blot and identified with SDS-PAGE. The result is consistent. The target protein can be specifically combined with His antibody. The antiserum prepared by Elisa is detected by Elisa method, the titer reaches 1:100 million, and the target strip in 55kD is obvious by Westernblot detection.
discuss
In this study, we first sequenced the synthesized Neurocan gene, showing that the sequence was correct, and the open reading frame and the enzyme cutting site were consistent with the design. Then, the plasmid PET30a (+) -Neurocan containing the Neurocan gene was transformed into the Escherichia coli BL21, the expression of the Neurocan protein was expressed, and the expressed protein was SDS- PAGE identification, the size of its molecular weight is in accordance with the size calculated by the base number of the base. Further Western blot identification is carried out, because the experiment has designed the His tag at the beginning of the Neurocan gene, so the anti His antibody is used. The result shows that the target protein can specifically bind to the His antibody, indicating that the target protein is the table of the Neurocan gene. The Neurocan gene was correctly identified by sequencing, suggesting that the prokaryotic expression of Neurocan protein was successful. The antiserum prepared by this protein was detected by Elisa, and the potency reached 1:100 million. Further detection by Western blot proved that the specificity was good and the prepared polyclonal antibody could be specifically combined with the Neurocan protein. Therefore, the antibody could be specifically combined with the Neurocan protein. The prokaryotic expression of Neurocan protein is successful, and the antibody produced by Neurocan protein can specifically bind to the Neurocan protein. It provides a useful reference for the further development of the DNA vaccine and the strategy for the repair of CNS after the injury.
【學位授予單位】：南方醫科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R651.3;R392

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