本文選題：人血白蛋白 + 分離純化　； 參考：《湖北中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：人血白蛋白(Human serum albumin,HSA)是由健康人的血漿經(jīng)低溫乙醇分離提取并經(jīng)病毒滅活后制成的生物制品,臨床上用于出血性、外傷性體克、燒傷、肝硬化伴腹水和水腫、惡性腫瘤、腎病水腫等。目前國內(nèi)企業(yè)采用低溫乙醇法壓濾工藝分離血漿蛋白,該工藝具有蛋白收率高,生產(chǎn)成本低,經(jīng)濟(jì)效益好,因而在工業(yè)化生產(chǎn)中大規(guī)模應(yīng)用。然而低溫乙醇法特異性不高,在生產(chǎn)的過程中不能完全除去雜蛋白成分,各企業(yè)的工藝參數(shù)也不盡相同,因此在終產(chǎn)品中會(huì)存在一定量的雜蛋白。據(jù)文獻(xiàn)表明,制劑中存在的這些雜蛋白是引起的臨床不良反應(yīng)的主要原因之一[1],不良反應(yīng)類型主要為全身過敏反應(yīng)和多器官損壞,包括過敏性休克、熱原樣反應(yīng)、腎臟損害等。因此探究血液制品中的未知蛋白可為臨床不良反應(yīng)提供參考,本課題旨在建立雜蛋白分離分析的方法并測(cè)定人血白蛋白制劑中的雜蛋白的組成,為各企業(yè)的生產(chǎn)工藝優(yōu)化提供了數(shù)據(jù)支持,同時(shí)為產(chǎn)品的一致性評(píng)價(jià)奠定基礎(chǔ)。為了初步了解各企業(yè)的雜蛋白含量和種類的差異,對(duì)五家企業(yè)近兩年的批簽發(fā)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,共獲得323批白蛋白制劑的純度數(shù)據(jù)。結(jié)果顯示,五家企業(yè)生產(chǎn)的白蛋白制劑純度范圍在96.2%~98.3%之間,企業(yè)間差異較大。其中A企業(yè)生產(chǎn)的制劑純度較為均一,提示生產(chǎn)工藝較為穩(wěn)定;B企業(yè)生產(chǎn)的制劑純度較其他四所企業(yè)低,顯示產(chǎn)品中存在一定量的未知蛋白。為了進(jìn)一步分析各企業(yè)制劑的蛋白組成異同,對(duì)白蛋白制劑進(jìn)行聚丙烯酰胺凝膠電泳(SDS-PAGE)分析,初步探究其組成成分的異同,結(jié)果顯示各企業(yè)間生產(chǎn)的白蛋白制劑差異較小,各企業(yè)制劑的雜蛋白有8~9條帶。鑒于醋酸纖維素薄膜電泳與SDS-PAGE方法的局限性,不能特異性地鑒定出雜蛋白成分,本研究嘗試建立未知蛋白譜鑒定方法,進(jìn)而確定各企業(yè)生產(chǎn)的人血白蛋白制劑的未知蛋白組成。在建立質(zhì)譜分析方法時(shí),首先對(duì)供試品濃度進(jìn)行選擇,再對(duì)質(zhì)譜鑒定參數(shù)進(jìn)行優(yōu)化。結(jié)果顯示,白蛋白濃度超過5mg/ml時(shí),超過儀器鑒定閾值,導(dǎo)致白蛋白主成分未能被成功鑒定,提示蛋白濃度不能過高。蛋白濃度在1mg/ml-2mg/ml時(shí),主成分白蛋白肽圖較為完整,但未知蛋白檢出效率低;對(duì)白蛋白肽圖中的肽段進(jìn)行b/y離子連續(xù)性分析發(fā)現(xiàn),蛋白鑒定得分大于150分,結(jié)果具有較高的可信度。以上結(jié)果提示主成分蛋白濃度過高,掩蓋未知蛋白的的信號(hào),未知蛋白的鑒定需要對(duì)未知蛋白進(jìn)一步分離。結(jié)合前期結(jié)果,對(duì)各企業(yè)的白蛋白中未知成分進(jìn)行分離純化、酶切方法的優(yōu)化,確立其分離純化方法,并分析各企業(yè)未知蛋白的種類、數(shù)量、分子量、等電點(diǎn)分布及其工藝相關(guān)性。采用陰離子交換柱與凝膠過濾層析柱分別對(duì)白蛋白進(jìn)行分離,結(jié)果顯示,使用陰離子交換層析只能得到主成分單一峰,而使用凝膠過濾層析得三組峰,因此采用凝膠過濾層析對(duì)白蛋白中位置組分進(jìn)行分離。采用胃蛋白酶和胰蛋白酶分別對(duì)各分離組分進(jìn)行酶切,結(jié)果顯示,胃蛋白酶酶切結(jié)果重復(fù)性較差且蛋白得分較低,而使用胰蛋白酶重現(xiàn)性良好,蛋白鑒定得分較高。對(duì)胰蛋白酶酶切效果進(jìn)行進(jìn)一步確證,結(jié)果發(fā)現(xiàn),三次平行實(shí)驗(yàn),共有11個(gè)蛋白被檢出,其中共有8個(gè)蛋白每次均檢出,說明方法穩(wěn)定性較好。最終采用胰蛋白酶對(duì)人血白蛋白中的未知蛋白成分進(jìn)行鑒定,結(jié)果顯示五家生產(chǎn)企業(yè)產(chǎn)品,除一家企業(yè)雜蛋白較少外,另外四家企業(yè)所有批次樣品均含有載脂蛋白a-Ⅱ(apolipoproteina-ii)、人結(jié)合珠蛋白(haptoglobin)、人血色素結(jié)合蛋白(hempoexin)、α-1b-糖蛋白(alpha-1b-glycoprotein)、α-2-hs-糖蛋白(alpha-2hs-glycoprotein),且略有差異。對(duì)未知蛋白等電點(diǎn)進(jìn)行分析發(fā)現(xiàn),未知蛋白等電點(diǎn)與白蛋白較接近,可能在乙醇共沉淀時(shí)與白蛋白共同析出,初步可以說明未知蛋白為工藝相關(guān)蛋白;對(duì)未知蛋白分子量進(jìn)行分析發(fā)現(xiàn),未知蛋白以多聚體的形式存在于白蛋白中。以上結(jié)果表明,在臨床應(yīng)用中,應(yīng)更加關(guān)注上述未知蛋白的過敏效應(yīng),在工業(yè)生產(chǎn)中,應(yīng)優(yōu)化生產(chǎn)工藝,盡可能去除無關(guān)蛋白,提高產(chǎn)品質(zhì)量。本課題建立的白蛋白制劑雜蛋白質(zhì)譜分析方法為探究臨床不良反應(yīng)提供了數(shù)據(jù)支持,為白蛋白制劑的一致性評(píng)價(jià)提供了新的方法,為血液制品的質(zhì)量控制奠定了基礎(chǔ)、提供了新思路。
[Abstract]:Human Albumin (Human serum albumin, HSA) is a biological product made from the plasma of healthy people and extracted by low temperature ethanol and inactivated by virus. It is used clinically for bleeding, traumatic body grams, burns, cirrhosis with ascites and edema, malignant tumor, nephrosis edema and so on. At present, the domestic enterprises adopt the low temperature ethanol filtration process to separate blood Plasma protein, which has high protein yield, low production cost and good economic benefit, is widely used in industrial production. However, the specificity of the low temperature ethanol method is not high. In the process of production, the protein components can not be completely removed, and the technological parameters of each enterprise are not the same. Therefore, there will be a certain amount of protein in the final products. It is shown in the literature that these proteins are one of the main causes of adverse reactions in the preparation of [1], and the types of adverse reactions are mainly systemic anaphylaxis and multiple organ damage, including anaphylactic shock, thermal response, kidney damage, etc. Therefore, the exploration of unknown proteins in the blood can provide a reference for adverse clinical reactions. The purpose of this study is to establish a method of separation and analysis of hetero protein and determine the composition of the heteroprotein in Human Albumin preparation. It provides data support for the optimization of the production process of various enterprises, and lays the foundation for the evaluation of the consistency of the products. In order to understand the differences in the content and types of the egg white in each enterprise, five enterprises have been used for the last two years. A total of 323 batch of albumin preparations were obtained by statistical analysis of the batch data. The results showed that the purity of the albumin preparation produced by the five enterprises was between 96.2%~98.3%, and the difference between enterprises was large. The purity of the preparation produced by the A enterprise was more uniform, indicating that the production process was more stable; the purity of the preparation produced by the B enterprise was more than that of it. In order to further analyze the protein composition and similarities and differences of various enterprise preparations, SDS-PAGE analysis of protein preparation was carried out to explore the similarities and differences of the composition of the components. The results showed that the differences in albumin preparation produced between enterprises were small, and the enterprises were small in different enterprises. The heteroproteins of the preparation have 8~9 bands. In view of the limitations of cellulose acetate membrane electrophoresis and the SDS-PAGE method, the hetero protein components can not be identified specifically. This study attempts to establish an unknown protein spectrum identification method to determine the unknown egg white composition of the Human Albumin preparation produced by the enterprises. The concentration of the sample was selected and the parameters of the mass spectrometry were optimized. The results showed that when the concentration of albumin exceeded 5mg/ml, the concentration of albumin could not be successfully identified and the protein concentration could not be successfully identified. The protein concentration was not too high. When the protein concentration was at 1mg/ml-2mg/ml, the protein peptide map of the principal component was more complete, but the unknown protein was detected. The b/y ion continuity analysis in the peptide map of the protein peptide map showed that the protein identification score was more than 150 points, and the results had high reliability. The above results suggested that the concentration of the principal component protein was too high to cover the signal of the unknown protein. The identification of the unknown protein should be further separated from the unknown protein. The separation and purification of the unknown components in the albumin of the enterprises, the optimization of the enzyme cutting method, the separation and purification method, and the analysis of the species, quantity, molecular weight, the distribution of the electric point and the correlation of the process of the unknown proteins of the enterprises were analyzed. The separation of white protein was carried out by the anion exchange column and the gel filtration column. The results showed that the use of the protein was used to separate the protein. The anion exchange chromatography can only get the single peak of the principal component, and the three groups of peaks are obtained by gel filtration chromatography. Therefore, the gel filtration chromatography is used to separate the position components in the white protein. The protease and trypsin are used to cut the separate components respectively. The results show that the results of the pepsin digestion are poor and the protein score is poor. It was low, and the trypsin reproducibility was good and the protein identification score was higher. The results of trypsin digestion were further confirmed. The results showed that 11 proteins were detected in three parallel experiments, of which 8 proteins were detected each time, indicating that the method was more stable with trypsin in the unknown Human Albumin. The protein components were identified, and the results showed that five production enterprise products, except one enterprise with less clutter, all the other four enterprise batch samples contain apolipoprotein a- II (apolipoproteina-ii), human globin (haptoglobin), human hemoglobin binding protein (hempoexin), alpha -1b- glycoprotein (alpha-1b-glycoprotein), and alpha -2-hs- sugar. Protein (alpha-2hs-glycoprotein), and slightly different. The analysis of the unknown protein and other electrical points found that the unknown protein and other electrical points are closer to the albumin, may precipitate together with the albumin when the ethanol is coprecipitation, and the unknown protein is a process related protein; the unknown protein molecular weight is found to be more than the unknown protein. The form of polymer exists in albumin. The above results show that in clinical application, we should pay more attention to the allergy effect of the above unknown protein. In industrial production, the production process should be optimized and the unrelated proteins should be removed as much as possible to improve the quality of the products. It provides data support, provides a new method for the consistency evaluation of albumin preparation, and lays a foundation for the quality control of blood products, and provides a new idea.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R927.1

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本文編號(hào)：1968635