本文關(guān)鍵詞： 丁肝抗原 HDV 血清學(xué)檢測(cè) 原核表達(dá)　出處：《中國(guó)疾病預(yù)防控制中心》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]Hepatitis D virus (HDV)可以引起丁型病毒性肝炎。HDV病毒的核蛋白即為丁肝抗原,可以刺激機(jī)體產(chǎn)生特異性的抗體。現(xiàn)在很多實(shí)驗(yàn)室使用重組生產(chǎn)的丁肝抗原作為標(biāo)準(zhǔn)抗原,對(duì)丁肝患者進(jìn)行血清學(xué)檢測(cè)。獲取高純度的丁肝抗原是本實(shí)驗(yàn)的第一個(gè)目的。并通過(guò)對(duì)表達(dá)純化資料的積累,為下一步的中試生產(chǎn)做準(zhǔn)備。當(dāng)前還沒(méi)有我國(guó)丁肝流行病學(xué)的準(zhǔn)確資料,主要是因?yàn)闆](méi)有統(tǒng)一的檢測(cè)標(biāo)準(zhǔn)和檢測(cè)試劑。使用本實(shí)驗(yàn)生產(chǎn)的丁肝抗原建立起特異性和敏感性均較好的血清學(xué)檢測(cè)方法是本實(shí)驗(yàn)的第二個(gè)目的。將其應(yīng)用于疾控工作,得到我國(guó)丁肝流行病學(xué)的準(zhǔn)確資料。 [材料和方法]本實(shí)驗(yàn)在Genbank數(shù)據(jù)庫(kù)獲取了HDV的序列信息,選擇HDV的S-HDag的編碼序列,在大腸桿菌偏好密碼子的基礎(chǔ)上,進(jìn)行了密碼子的替換。預(yù)測(cè)了該抗原mRNA的二級(jí)結(jié)構(gòu),對(duì)影響表達(dá)的序列通過(guò)簡(jiǎn)并密碼子進(jìn)一步優(yōu)化后,人工合成該基因。通過(guò)基因工程技術(shù),雙酶切后,連接至pET43.1.a表達(dá)載體,在大腸桿菌BL21(DE3)工程菌株中進(jìn)行表達(dá)。通過(guò)控制表達(dá)條件,最終確定的表達(dá)程序?yàn)椋簶?biāo)準(zhǔn)轉(zhuǎn)化程序獲取的單克隆接種5m1小試管,37攝氏度過(guò)夜培養(yǎng)后,1：50稀釋接種于250m1錐形瓶中,37攝氏度生長(zhǎng)至OD值約為0.8,使用誘導(dǎo)劑為ITPG,終濃度為1mM/L,37攝氏度下誘導(dǎo)3h,最終獲得HDV抗原的可溶性表達(dá)。使用的培養(yǎng)基為L(zhǎng)B培養(yǎng)基,選擇性的抗性為氨芐青霉素,使用終濃度為50mM/L。 獲得的大腸桿菌菌體,使用超聲破碎儀,完全破碎菌體,在10000rpm下離心,保留上清,在1M/L的NaCl條件下,利用表達(dá)目的蛋白的His-tag,在鎳離子和咪唑的作用下,通過(guò)親和層析初步獲得了目的蛋白,后進(jìn)一步經(jīng)過(guò)離子交換層析和分子篩層析,最終獲得了高純度的蛋白,符合血清學(xué)檢測(cè)試劑盒應(yīng)用的標(biāo)準(zhǔn)。 將本實(shí)驗(yàn)獲取的目的蛋白包被ELISA板,包被的條件是在pH9.5的包被碳酸緩沖液作用下,每孔2-4ng,4攝氏度過(guò)夜,組裝成檢測(cè)試劑盒,通過(guò)間接法測(cè)定標(biāo)本中的IgM抗體,根據(jù)結(jié)果對(duì)其進(jìn)行判定。對(duì)來(lái)自丁肝血清盤(pán)的標(biāo)本和臨床收集的血清標(biāo)本進(jìn)行檢測(cè),該方法的特異性和敏感性均較好。 [結(jié)論]本實(shí)驗(yàn)獲得了具有抗原活性的目的蛋白,建立了血清學(xué)檢測(cè)方法。通過(guò)基因工程技術(shù),表達(dá)目的蛋白,為下一步的中試生產(chǎn)做好了技術(shù)準(zhǔn)備。低成本的優(yōu)勢(shì)和良好的效果可以為該檢測(cè)方法的市場(chǎng)化做鋪墊,進(jìn)而確定統(tǒng)一的標(biāo)準(zhǔn)和試劑,從而得到我國(guó)準(zhǔn)確的流行病學(xué)資料。
[Abstract]:[Objective] the nucleoprotein of Hepatitis D virus can cause hepatitis D. the nucleoprotein of HDV is liver D antigen. It stimulates the body to produce specific antibodies. Many laboratories now use recombinant hepatitis D antigens as standard antigens. It is the first aim of this experiment to obtain high purity of hepatitis D antigen by serological examination in patients with hepatitis D. and through the accumulation of purified data of expression. To prepare for the next pilot-scale production. At present, there are no accurate data on the epidemiology of hepatitis D in China. The main reason is that there is no uniform detection standard and reagent. It is the second purpose of this experiment to establish a specific and sensitive serological detection method using the antigens produced in this experiment. Control work. The accurate data of hepatitis D epidemiology in China were obtained. [Materials and methods] the sequence information of HDV was obtained from Genbank database and the coding sequence of S-HDag of HDV was selected based on the preference codon of Escherichia coli. The secondary structure of the antigen mRNA was predicted. After further optimization of the sequence affecting the expression by degenerate codon, the gene was synthesized artificially. Ligated to pET43.1.a expression vector and expressed in Escherichia coli BL21DE3 engineering strain by controlling the expression conditions. The final expression procedure was as follows: 1: 50 was diluted into 250m1 conical bottle after overnight culture with monoclonal inoculation of 5m1 small test tube and 37 degrees Celsius by standard transformation procedure. 37 degrees Celsius grew to an OD value of about 0.8, the inducer was ITPG.The final concentration was 1 mm / L ~ (37 鈩，

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