TW202140780A - 新穎的具有改善的穩定性的玻尿酸酶變異體及含有其的醫藥組合物 - Google Patents

新穎的具有改善的穩定性的玻尿酸酶變異體及含有其的醫藥組合物 Download PDF

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TW202140780A
TW202140780A TW110102662A TW110102662A TW202140780A TW 202140780 A TW202140780 A TW 202140780A TW 110102662 A TW110102662 A TW 110102662A TW 110102662 A TW110102662 A TW 110102662A TW 202140780 A TW202140780 A TW 202140780A
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朴淳宰
鄭惠信
李承柱
金奎完
宋炯枏
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南韓商阿特根公司
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Abstract

揭露了新穎的PH20變異體或其片段，具有改善熱穩定性及酵素活性的人類玻尿酸酶，其中玻尿酸酶為水解玻尿酸的酵素，尤其是關於在具有SEQ ID NO: 3之胺基酸序列的變體中包含一或多個胺基酸殘基取代的PH20變異體或其片段，其中一或多胺基酸殘基在N端及/或C端更被選擇性地刪除。

Description

新穎的具有改善的穩定性的玻尿酸酶變異體及含有其的醫藥組合物
本發明是關於新穎人類PH20變異體或其片段，其相較於人類玻尿酸酶（hyaluronidase）具有增加的酵素活性及熱穩定性，其中所述玻尿酸酶為一種水解玻尿酸（hyaluronic acid）的酵素。本發明尤其是關於在具有SEQ ID NO: 3之胺基酸序列的玻尿酸酶變異體中包含一或多胺基酸殘基取代（substitution）、刪除（deletion）及/或插入（insertion）的PH20變異體或其片段，且其中一或多胺基酸殘基選擇性地被從N端及/或C端刪除，用於製造所述PH20變異體或其片段的方法，及包含所述PH20變異體或其片段的醫藥組成物。
人類皮膚是由表皮、真皮及皮下脂肪層組成，且皮膚中有六種醣胺聚多醣（glycosaminoglycan）。這些醣胺聚多醣包含玻尿酸、硫酸軟骨素（chondroitin sulfate）、硫酸皮膚素（dermatan sulfate）、硫酸乙醯肝素（heparan sulfate）、肝素（heparin）及硫酸角質素（keratin sulfate）。
這些醣胺聚多醣是由重複的雙醣糖單元（disaccharide sugar unit）組成。各醣胺聚多醣中之重複的雙醣糖單元的數量有所不同，但其差異量的範圍是從幾百個到幾千個。在這些醣胺聚多醣中，玻尿酸在皮膚中的含量佔人體的一半以上。玻尿酸是由細胞膜中的玻尿酸合成酶所合成，為單獨存在而不與蛋白多醣（proteoglycans）結合，且為唯一不具有硫酸鹽基團（sulfate group）的醣胺聚多醣。其他醣胺聚多醣與蛋白多醣結合，且具有硫酸鹽基團。玻尿酸由透過β-1,4及β-1,3鏈交替連接的葡萄糖醛酸（glucuronic acid）及N-乙醯葡萄胺糖（N-acetylglucosamine）組成，且由這些雙醣的5000個重複單元構成。已知的是，人體中每天約有三分之一（5 g）的玻尿酸被降解。
玻尿酸酶為降解細胞外基質（extracellular matrix）中的玻尿酸的酵素。六個已知的人類玻尿酸酶基因：Hyal1、Hyal2、Hyal3、Hyal4、HyalPS1及PH20/SPAM1。人類的Hyal1及Hyal2基因表現在多數組織中。PH20/SPAM1（以下簡稱PH20）表現在精子細胞膜（sperm plasma membrane）及頂體膜（acrosomal membrane）。然而，HyalPS1因其為假基因（pseudogene）而未被表現。取決於玻尿酸的切割方法，玻尿酸酶被分為三類：利用水分子（H2 O）切割N-乙醯葡萄胺糖與葡萄糖醛酸之間的β-1,4鏈的酵素（EC 3.2.1.35）；利用水分子切割N-乙醯葡萄胺糖與葡萄糖醛酸之間的β-1,3鏈的酵素（EC 3.2.1.36）；以及不使用水分子切割β-1,4鏈的細菌型（bacterial）玻尿酸酶（EC 4.2.99.1）。
Hyal1的催化胺基酸（catalytic amino acid）為透過受質輔助催化（substrate-assisted catalysis）水解玻尿酸的D129及E131。Hyal1在酸性pH值3至4下表現出最佳活性，在pH值4.5或以上不具有活性。相較於Hyal1，PH20在廣泛的pH值範圍3至8皆表現出活性。
Arming等人辨識出PH20的催化胺基酸為D111及E113（Arming等人，1997）。Arming等人指定Leu為PH20的第一個胺基酸，其中訊息肽（signal peptide）等被移除，並因此包含訊息肽的PH20的催化胺基酸分別對應到D146及E148。
玻尿酸酶水解玻尿酸，進而降低細胞外基質中玻尿酸的黏性（viscosity），並增加其向組織（皮膚）的滲透性。皮膚的皮下區域具有中性的pH值約7.0至7.5。因此，在玻尿酸酶的各種類型中，PH20在臨床上被廣泛地使用（Bookbinder等人，2006）。在PH20在臨床上被使用的例子中，PH20在眼科手術中是作為眼部鬆弛劑及麻醉添加劑，且亦與皮下注射的抗體治療劑被共同施用（Bookbinder等人，2006）。此外，基於玻尿酸在腫瘤中會過度表現的特性，PH20用於水解腫瘤細胞的細胞外基質中的玻尿酸，進而增加抗腫瘤治療劑進入腫瘤細胞的機會。此外，其亦用於促進人體組織中過量存在的體液及血液的吸收。
PH20是由Lathrop等人首次在天竺鼠的精液中被辨識出來，也已知在不同物種的精子中表現。人類PH20基因是由Lin等人及Gmachl等人選殖。人類的PH20具有SEQ ID NO: 1的胺基酸序列，其由509個胺基酸殘基組成，且表現出與60%的天竺鼠的PH20基因的一致性（identity）。人類的PH20酵素係由SPAM1（sperm adhesion molecule-1）基因編碼，且PH20的Ser490在精子細胞膜表面上及頂體膜中以與多醣磷脂肌醇（glycosylphosphatidylinositol，GPI）結合的形式存在。當精子穿過卵子富含玻尿酸的卵丘層（cumulus layer）滲透進卵子時，精子使用PH20以水解玻尿酸。PH20的含量相當於精子中蛋白質含量的1%或更少，且具有六個N-醣基化位點（N-glycosylation site）（N82、N166、N235、N254、N368及N393）。
目前可商購的PH20是透過從牛或綿羊的睪丸中萃取而得的。其例子包含Amphadase®（胎牛玻尿酸酶（bovine hyaluronidase））及Vitrase®（綿羊玻尿酸酶（sheep hyaluronidase））。
胎牛睪丸玻尿酸酶（BTH）是透過在轉譯後修飾（post-translational modification）期間，從胎牛野生型PH20在C端移除訊息肽及56個胺基酸而得。BTH也是一種醣蛋白，且基於包含胺基酸在內的總成分，其甘露糖（mannose）含量為5%，葡萄糖胺（glucosamine）含量為2.2%。當動物來源的玻尿酸酶被以高劑量重複地施用於人體時，可以產生中和抗體。由於動物來源的玻尿酸酶除了PH20外還含有其他生物材料，當被施用於人體時其可能導致過敏反應（Bookbinder等人，2006）。尤其，由於擔心瘋牛病，從牛萃取出的PH20的製造及使用可能受到限制。為了克服這個問題，已經對人類PH20的重組蛋白進行很多研究。
人類PH20的重組蛋白已被發現在酵母（P. pastoris ）、DS-2昆蟲細胞及動物細胞中表現。在昆蟲細胞及酵母中製造的PH20重組蛋白的N-醣基化在轉譯後修飾期間的形態與人類PH20不同。
玻尿酸酶，其蛋白質結構已辨識為Hyal1（PDB ID: 2PE4）（Chao等人，2007）及蜂毒玻尿酸酶（PDB ID: 1FCQ, 1FCU, 1FCV）。Hyal1由兩個結構域（domain）組成，分別為催化結構域及表皮生長因子樣結構域（EGF-like domain）。催化結構域為(β/α)8 形式，其中表徵蛋白質二級結構的α螺旋（α-helix）及貝他股（beta-strand）各重複八次（Chao等人，2007）。在其中Hyal1的C端以不同剪接方式的變體中，表皮生長因子樣結構域完全保留。Hyal1及PH20的胺基酸序列有35.1%的一致性，且PH20的蛋白質結構尚未被發現。
人類PH20的蛋白質重組是由Halozyme Therapeutic, Inc.開發，且已被以商品名稱Hylenex®售出（Bookbinder等人，2006；Frost，2007）。
當PH20的催化胺基酸D146及E148分別被變異為天冬醯胺素（asparagine）（D146N）及麩醯胺（glutamine）（E148Q）時不存在酵素活性（Arming等人，1997）。此外，當PH20的R246被麩醯胺酸（glycine）替代時，酵素活性降低90%，而當E319被麩醯胺酸替代時，酵素活性消失。相較於野生型PH20，在C端移除36個胺基酸（截斷胺基酸474-509）的PH20的變異體，其酵素活性降低了75%。此突變體不是在細胞外分泌的，而是存留在希拉細胞（HeLa cell）中。在C端移除134個胺基酸的PH20的突變體不具有酵素活性，且不是在細胞外分泌的。根據Frost等人，PH20的C端的477-483區對於可溶性表達（soluble expression）是不可少的（Frost，2007）。完整長度的PH20（1-509）或在C端的467位置被截斷的PH20變異體的活性僅為在C端的477-483其中一個位置被截斷的PH20變異體的10%（Frost，2007）。
重組的PH20在醫學上被用為載體以促進藥物在皮下的傳遞，以降低患有眼科疾病的病人的眼壓、以在手術後延緩狹窄（stenosis）、作為分散劑（dispersant）以促進例如癌症等疾病中的化療藥物的活性、在手術中作為輔助治療劑等。
特別是在蛋白質藥物的情況下，近來，開發出了濃度範圍從每1 毫升（mL）有幾十毫克到幾百毫克不等的高劑量產品，且因此重組PH20作為載體以促進如蛋白質藥物的皮下傳遞的應用亦增加。蛋白質藥物可能有因其高濃度而造成黏度及蛋白質聚集（aggregation）程度增加進而導致的低物理穩定性的問題。此外，蛋白質聚集是不可逆的，且少量的蛋白開始聚集且聚集會形成更大的塊（clump）（Schön等人，2015）。亦即，合併施用的重組PH20會發生聚集，進而降低蛋白質藥物的穩定性。
同時，在熱穩定性及表達程度上，傳統的重組PH20仍是不足的。因此，產業上對具有進一步改善的生物和物理化學性質的重組玻尿酸酶的需求很大。
參考資料 Arming, S., Strobl, B., Wechselberger, C., and Kreil, G. (1997). In-vitro mutagenesis of PH-20 hyaluronidase from human sperm. Eur. J. Biochem.247 , 810-814. Bookbinder, L.H., Hofer, A., Haller, M.F., Zepeda, M.L., Keller, G.A., Lim, J.E., Edgington, T.S., Shepard, H.M., Patton, J.S., and Frost, G.I. (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J. Control. Release114 , 230-241. Chao, K.L., Muthukumar, L., and Herzberg, O. (2007). Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry46 , 6911-6920. Frost, G.I. (2007). Recombinant human 玻尿酸酶hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin. Drug Deliv.4 , 427-440. Schön, A., Clarkson, B.R., Siles, R., Ross, P., Brown, R.K., Freire, E. (2015) Denatured state aggregation parameters derived from concentration dependence of protein stability. Anal. Chem.488 , 45-50 WO 2020/022791A (2020. 1. 30.)
因此，鑒於上述問題而完成本發明，本發明一目的在於提供一種PH20變異體或其片段，相較於野生型PH20，尤其成熟的野生型PH20，其熱穩定性、酵素活性及表現程度會受到改善。
本發明另一目的在於提供一種組成物，其中組成物包含PH20變異體或其片段。
根據本發明一態樣，上述及其他目的能透過提供PH20變異體或其片段來達成，其中PH20變異體或其片段在具有SEQ ID NO: 3之胺基酸序列之玻尿酸酶變異體中包含一或多個胺基酸殘基取代、刪除及/或插入，其中在N端或在C端之一或多個胺基酸殘基選擇性刪除。
根據本發明另一態樣，提供有一種用於治療癌症之組成物及使用其治療癌症之方法，其中組成物包含PH20變異體或其片段。
發明功效
當在CHO（ExpiCHO）細胞中表現時，根據本發明之PH20變異體或其片段具有提升的蛋白質表現程度，且蛋白質聚集溫度（aggregation temperature）增加約4到11.5℃，故根據本發明之PH20變異體或其片段被有效地生產，且相較於成熟的野生型PH20具有更高的熱穩定性。
此外，如作為量測玻尿酸酶的活性的多個測試中的一者之受質膠測定（substrate-gel assay）的結果，根據本發明的PH20變異體或其片段具有改善蛋白質重新摺疊（protein refold）的效果，因此與成熟的野生型PH20相比，其復性（re-nature）速度更快，且不論C端的切割位至，仍能保留原始的酵素活性。
再者，根據本發明之PH20變異體或其片段具有低免疫原性（immunogenicity），而使得其能重複地施用於人體。
除非另有定義，否則本文所使用之所有技術及科學術語具有相同於本發明所屬領域中具有通常知識者所理解的含意。一般而言，本文所使用之命名為本領域習知的且為通常所使用的。
在本發明中，當被描述為基於野生型PH20時，各變異體之胺基酸殘基的位置係參考自SEQ ID NO: 1之胺基酸序列被描述，當被描述基於具有SEQ ID NO: 3之PH20變異體時，各變異體之胺基酸殘基的位置係參考自SEQ ID NO: 3之胺基酸序列。
本案發明人透過先前研究發現一種玻尿酸酶PH20變異體，並提出了關於此發現的專利申請（請見WO 2020/022791A），此玻尿酸酶PH20變異體在具有SEQ ID NO: 1之胺基酸序列之野生型PH20（較佳為成熟的野生型PH20）中在對應α螺旋域及/或其連接鏈域（較佳為α螺旋8域（S347至C381）及/或α螺旋7及α螺旋8之間的連接鏈域（A333至R346））的區域包含一或多個胺基酸殘基取代，其中可選地，一或多個N端及/或C端胺基酸殘基被選擇性切割或刪除，相較於習知的野生型PH 20或其片段，此玻尿酸酶PH20變異體展現出優越的功效。
如本文所使用之術語「成熟的野生型PH20」係指由SEQ ID NO: 1之L36至Y482或L36至S490之胺基酸殘基組成之蛋白質，所述的胺基酸殘基缺乏在野生型PH20之SEQ ID NO: 1之胺基酸序列中形成訊息肽之M1至T35，以及與PH20之實質酵素功能無關的N483至L509或A491至L509的其中一者。
具體而言，本案發明人透過先前研究發現，當在具有SEQ ID NO: 1之胺基酸序列的野生型PH20中對應T341至I361之胺基酸位置（其為α螺旋8域（S347至C381）及/或α螺旋7及α螺旋8之間的連接鏈域（A333至R346）的一部分）以對應具有SEQ ID NO: 2之序列的野生型Hyal1的胺基酸殘基取代時，表現效率及酵素活性會受到改善，並且在N端及C端之部分胺基酸序列刪除的片段亦展現出優越的表現效率及高酵素活性。
表1：野生型PH20及野生型Hyal1之胺基酸序列
野生型PH20之胺基酸序列（SEQ ID NO: 1）   MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL
野生型Hyal1之胺基酸序列（SEQ ID NO: 2）   MAAHLLPICALFLTLLDMAQGFRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEFKCRCYPGWQAPWCERKSMW
作為其持續研究的結果，本案發明人發現儘管具有SEQ ID NO: 3之序列的變異體包含胺基酸殘基之額外的取代、刪除及/或插入，以及更可選地包含在N端及/或C端之一或多個胺基酸殘基的刪除，但具有SEQ ID NO: 3之序列的變異體相較於野生型PH20仍展現出優異的表現效率、高酵素活性以及顯著改善的蛋白質聚集溫度（aggregation temperature，Tagg ），其中具有SEQ ID NO: 3之序列的變異體係藉由將對應具有SEQ ID NO: 1之胺基酸序列的野生型PH20之T341至I361之胺基酸區域以對應具有SEQ ID NO: 2之野生型Hyal1之胺基酸序列取代來建構而得。基於此發現，完成本發明。
具有SEQ ID NO: 3之序列的變異體係藉由將15個胺基酸殘基取代來建構而得，亦即，在具有SEQ ID NO: 1之胺基酸序列的野生型PH20中之T341S、L342W、S343E、I344N、M345T、S347T、M348K、K349E、L352Q、L353A、L354I、D355K、N356E、E359D及I361T。
在此方面，根據本發明之PH20變異體或其片段在具有SEQ ID NO: 3之胺基酸序列的PH20變異體中包含一或多個胺基酸殘基的取代、刪除及/或插入，並可選地在N端及/或C端包含一或多個胺基酸殘基的刪除。
如上所述，具有SEQ ID NO: 3之胺基酸序列的變異體係野生型PH20之T341至I361之胺基酸殘基被野生型Hyal1之對應的胺基酸殘基取代之變異體（請見表2）。在先前研究中，在N端及C端包含胺基酸殘基刪除之具有SEQ ID NO: 3之胺基酸序列的變異體或其片段被認為是在活性及穩定性上優於野生型PH20之變異體。
表2，野生型PH20之位置T341至I361的胺基酸殘基被Hyl1之對應的胺基酸殘基取代之PH20變異體的胺基酸序列 （SEQ ID NO: 3）
MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG SWENTRTKESCQAIKEYMDTT LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL
具體而言，根據本發明之PH20變異體或其片段可包含一或多個突變，較佳為在SEQ ID NO: 3之胺基酸序列中之一或多個胺基酸殘基的取代、刪除及/或插入，並具有較野生型PH20更高的蛋白質聚集溫度（Tagg ），其中蛋白質聚集溫度為表示蛋白質穩定性的指標。此外，根據本發明之PH20變異體不包含SEQ ID NO: 1之野生型PH20。
如本文所使用之術語「PH20變異體」旨在包含一種變異體，其不僅具有一或多個胺基酸殘基的突變（較佳為一或多個胺基酸殘基的取代、刪除及/或插入），更具有在其N端或C端一或多個胺基酸殘基刪除與胺基酸殘基的取代、刪除及/或插入，且其用法與「PH20變異體或其片段」的含意實質上相同。
較佳地，依據本發明的PH20變異體包含胺基酸殘基在具有SEQ ID NO: 3之胺基酸序列的變異體中之選自由R39、D65至L68、N82、T84、I102至I105、T132至Y134、N166、L179至T182、T185至K187、V241至K244、N266至Q269、P271、V272、K290至P292、Q311至K314、G340至N363、L441、S442、D451至D453、D461、V463及D461至V463組成之群組之至少一位置包含胺基酸殘基的取代、刪除及/或插入，且具有較野生型PH20高的蛋白質聚集溫度（Tagg ）。
依據本發明的PH20變異體PH20在SEQ ID NO: 3之胺基酸序列中可以在20或更少（較佳為17或更少，更理想為15或更少）的胺基酸位置包含突變，但不限於此。
較佳地，依據本發明的PH20變異體或其片段包含在具有SEQ ID NO: 3之胺基酸序列的變體中，選自由R39K、D65A、E66A、P67A、L68A、N82A、T84N、I102A、D103A、S104A、S104N、I105A、I105Q、T132A、T132S、F133A、Y134A、N166A、N166K、L179A、L179S、L179I、L179F、S180T、S180A、L181A、L181M、T182A、T185A、E186A、E186D、K187A、V241A、E242A、I243A、K244A、N266A、T267A、Q268A、Q268D、Q268I、Q268N、Q269A、P271A、V272A、K290A、I291A、I291G、I291L、P292A、P292D、Q311A、V312A、L313A、L313P、L313M、K314A、G340Q、S341H、S341D、S341T、W342I、W342D、W342H、W342L、E343V、E343S、E343Y、E343Q、N344F、N344I、T345E、T345K、T345S、R346M、R346F、R346L、R346T、R346S、R346A、T347Q、T347E、T347V、T347W、T347H、T347S、K348Q、K348F、K348D、K348T、K348E、K348M、E349L、E349W、E349A、S350Q、S350I、S350D、S350T、S350E、S350N、Q352E、Q352G、Q352Y、Q352W、Q352T、A353E、A353Y、A353H、A353K、I354E、I354Q、I354S、I354V、I354A、I354N、I354T、I354R、I354W、I354L、K355Q、K355H、K355D、E356M、E356F、E356I、E356L、E356Q、E356V、E356D、 Y357W、Y357F、M358V、M358R、M358Y、M358L、D359K、D359V、D359Y、D359Q、D359T、D359S、D359E、T360Y、T360R、T360L、T360D、T360S、T361M、T361E、T361H、T361L、T361D、T361I、L362A、N363M、N363E、L441A、S442A、D451A、D451S、T452A、T452D、T452H、T452K、T452G、T452P、T452M、T452F、D453A、D461R、D461A、G462A、V463Y以及V463A組成之群組之至少一者的胺基酸殘基取代，但不限於此。
在本發明中，由胺基酸殘基碼的一個字母與數字組成的描述（如S341），是指在SEQ ID NO: 1或SEQ ID NO: 3的胺基酸序列中在每個位置的胺基酸殘基。
例如，「S341」是指SEQ ID NO: 3之胺基酸序列中在位置341的胺基酸殘基為絲胺酸（serine），而「S341H」是指在SEQ ID NO: 3的位置341的絲胺酸被組胺酸（histidine）取代。
依據本發明的PH20變異體或其片段被解釋為包含變異體或其片段，且其中在特定胺基酸殘基位置的胺基酸殘基被保守地取代。
如本文所使用之術語「保守取代」是指PH20變異體的修飾，其是關於以其他具有相似生化特性的胺基酸取代一或多個胺基酸，其中所述具有相似生化特性是指不會造成PH20變異體的生物或生化功能喪失。
術語「保守胺基酸取代」是指以具有相似側鏈的另一胺基酸殘基取代胺基酸殘基。具有相似側鏈的胺基酸殘基家族已被定義且並且在本發明所屬領域中是為人所知的。這家些族包含具有基本側鏈的胺基酸（如離胺酸（lysine）、精胺酸（arginine）及組胺酸（histidine））、具有酸側鏈的胺基酸（如天冬醯胺酸（aspartic acid）及麩醯胺酸（glutamic acid））、具有不帶電荷的極性側鏈的胺基酸（如天門冬素、麩醯胺、絲氨酸、蘇氨酸（threonine）、酪胺酸（tyrosine）及半胱氨酸（cysteine））、具有非極性側鏈的胺基酸（如甘胺酸、丙氨酸（alanine）、纈氨酸（valine）、白氨酸（leucine）、異白胺酸（isoleucine）、脯氨酸（proline）、苯丙氨酸（phenylalanine）、甲硫氨酸（methionine）及色氨酸（tryptophan））、具有貝他（beta）側鏈的胺基酸（如蘇氨酸、纈氨酸及異白胺酸）以及具有芳香族側鏈的胺基酸（如酪氨酸、苯丙氨酸、色氨酸及組胺酸。
本發明的PH20變異體或其片段被發現儘管具有保守的胺基酸取代，仍保有其活性（activity）。
此外，依據本發明的PH20變異體或其片段被解釋為包含具有與依據本發明的PH20變異體或其片段實質上相同的功能及/或效果的PH20變異體及其片段，且具有至少80%或85%與依據本發明的PH20變異體或其片段同源（homology）的胺基酸序列，較佳為至少90%同源，更佳為至少95%同源，且最佳為至少99%同源。
依據本發明的PH20變異體或其片段具有上升的表現程度及蛋白重新摺疊率，且進而具有較成熟的野生型PH20更高的熱穩定性。此外，儘管熱穩定性的上升，PH20變異體的酵素活性仍大於或相近於野生型PH20的酵素活性。
同時，雖然在C端具有切割的成熟的野生型PH20已知為具有降低的酵素活性，依據本發明的PH20變異體儘管在C端的一或多個胺基酸殘基，及/或在N端的1到7（較佳為1到5）的胺基酸殘基被切割及刪除，仍展現了相近或增加的酵素活性及表現效率，以及因較快速的蛋白重新摺疊率及其熱穩定性而有高的蛋白質聚集溫度（Tagg ）。
據此，依據本發明的PH20變異體或其片段的特徵在於其包含一或多個胺基酸突變（較佳為在具有SEQ ID NO: 3之胺基酸序列等的變異體中的一或多個胺基酸取代、刪除及/或插入）及在N端及/或C端的一或多個胺基酸殘基被額外刪除，但不限於此。
在一實施例中，依據本發明的PH20變異體或其片段可以為其中切割是發生在自從N端的M1至P42組成的群組選擇的胺基酸殘基之前，較佳是在SEQ ID NO: 3之胺基酸序列中N端的L36、N37、F38、R39、A40、P41或P42胺基酸殘基之前，以使N端的一或多個胺基酸殘基被刪除，及/或切割是發生在自從C端的V455到L509組成的群組選擇的胺基酸殘基之後，較佳是在自V455到S490組成的群組選擇的胺基酸殘基之後，更佳是在自V455、D456、C458、D461、C464、I465、D466、A467、F468、K470、P471、P472、M473、E474、T475、E476、P478、I480、Y482、A484、P486、T488或S490組成的群組選擇的胺基酸殘基之後，以使在C端的一或多個胺基酸殘基被刪除。
所述「切割是發生在自從N端的M1至P42組成的群組選擇的胺基酸殘基之前」是指緊接在從在N端的M1至P42中所選胺基酸殘基之前的一部份的胺基酸殘基被切割並刪除。所述「切割發生在M1之前」是指沒有切割是發生在N端。
舉例來說，所述「切割發生在胺基酸殘基L36、N37、F38、R39、A40、P41或P42之前」是指在依據本發明的SEQ ID NO: 3之胺基酸序列中，從M1到T35的所有緊接在L36之前的胺基酸殘基、從M1到L36的所有緊接在N37之前的胺基酸殘基、從M1到N37的所有緊接在F38之前的胺基酸殘基、從M1到F38的所有緊接在R39之前的胺基酸殘基、從M1到R39的所有緊接在A40之前的胺基酸殘基、從M1到A40的所有緊接在P41之前的胺基酸殘基或從M1到P41的所有緊接在P42之前的胺基酸殘基被切割及刪除。
此外，所述「切割是發生在自從C端的V455到L509組成的群組選擇的胺基酸殘基之後」是指緊接在從在C端的V455到L509中所選胺基酸殘基之後的一部份的胺基酸殘基被切割及刪除。
舉例來說，所述「切割發生在C端的胺基酸殘基V455、D456、C458、D461、C464、I465、D466、A467、F468、K470、P471、P472、M473、E474、T475、E476、P478、I480、Y482、A484、P486、T488或S490之後」是指在依據本發明的SEQ ID NO: 3之胺基酸序列中，在胺基酸殘基V455、D456、C458、D461、C464、I465、D466、A467、F468、K470、P471、P472、M473、E474、T475、E476、P478、I480、Y482、A484、P486、T488或S490之後的胺基酸殘基被切割，或緊接在所選的胺基酸殘基之後到L509的所有胺基酸殘基被刪除。
較佳地，根據本發明的新穎的PH20變異體或其片段的特徵在於，其包含在具有SEQ ID NO: 3之胺基酸序列的變體中一或多個位置的胺基酸殘基取代、刪除或插入、在N端在F38之前的截斷及在C端在F468之後的截斷。
更佳地，根據本發明的新穎的PH20變異體或其片段可以包含從由SEQ ID NOS: 163至316的胺基酸序列組成的群組中選擇的胺基酸序列，但不限於此。
建構於依據本發明特定實施例中的在PH20變異體中被取代或切割的胺基酸的序列如表6所示。
此外，在本發明中，嘗試使用在動物細胞中高度表現的其他訊息肽以增加重組PH20蛋白質的表現，而不是使用PH20原始的訊息肽。
因此，在另一實施例中，根據本發明的新穎的PH20變異體可以如下表3所示為其中N端更包含具有SEQ ID NO: 4的胺基酸序列MATGSRTSLLLAFGLLCLPWLQEGSA的人類生長賀爾蒙訊息肽、具有SEQ ID NO: 5的胺基酸序列MKWVTFISLLFLFSSAYS的人類血清白蛋白訊息肽，或具有SEQ ID NO: 6的胺基酸序列MAAHLLPICALFLTLLDMAQG的人類Hyal1訊息肽，代替由M1到T35組成的野生型PH20的訊息肽，但不限於此。
所述「代替由M1到T35組成的野生型PH20的訊息肽」是指SEQ ID NO: 3之胺基酸序列中訊息肽被部分或完全刪除的情況，且因此不會執行其功能。此外，上述說明是欲包含N端的一部份被進一步刪除的情況，例如，發生了切割發生在N37、F38、R39、A40、P41或P42殘基之前的情況，使N端的額外刪除與野生型PH20的訊息肽的刪除一起發生。
表3，根據本發明之訊息肽序列
  胺基酸序列 SEQ ID NO.
人類生長賀爾蒙 MATGSRTSLLLAFGLLCLPWLQEGSA 4
人類血清白蛋白 MKWVTFISLLFLFSSAYS 5
人類Hyal1 MAAHLLPICALFLTLLDMAQG 6
另一方面，本發明涉及一種用於治療癌症的組合物及使用所述組合物以治療癌症的方法，其中所述組合物包含根據本發明的新穎的PH20變異體或其片段。
可由根據本發明的新穎的PH20變異體或其片段治療的癌症（cancer）或癌（carcinomas）並不特別限制，但包含實質固態瘤與血癌。所述癌症可以選自由皮膚癌，例如黑素瘤、肝癌、肝細胞癌、胃癌、乳癌、肺癌、卵巢癌、支氣管癌、鼻咽癌、喉癌、胰腺癌、膀胱癌、大腸直腸癌、結腸癌、子宮頸癌、腦癌、前列腺癌、骨癌、甲狀腺癌、副甲狀腺癌、腎癌、食道癌、膽管癌、睾丸癌、直腸癌、頭頸癌、輸尿管癌、骨肉瘤、 神經細胞瘤、肉瘤、橫紋肌肉瘤、星形細胞瘤、神經母細胞瘤和神經膠質瘤等組成的群組，但不限於此。較佳地，可由根據本發明的組合物治療的癌症可以選自由大腸直腸癌、乳癌、肺癌及腎癌組成的群組，但不限於此。
本發明的組成物可以為醫藥組成物。醫藥組成物可以更包含醫學上可接受的組成物。所述組成物可以含有自由乳糖、右旋糖（dextrose）、蔗糖、山梨醇（sorbitol）、甘露醇、澱粉、阿拉伯膠（gum acacia）、磷酸鈣、藻酸鹽（alginate）、明膠、矽酸鈣、微晶纖維素（microcrystalline cellulose）、聚乙烯吡咯烷酮（polyvinylpyrrolidone）、纖維素、水、糖漿、甲基纖維素、甲基羥基苯甲酸酯（methylhydroxybenzoate）、丙基羥基苯甲酸酯（propylhydroxybenzoate）、滑石（talc）、硬脂酸鎂（magnesium stearate）及礦物油組成的群組選擇之一或多個物質，其中上述組成物可以含有的物質常用於製備藥物，但不限於此。此外，醫藥組成物更可以含有由稀釋劑、賦形劑、潤滑劑、潤濕劑、甜味劑、芳烴、乳化劑、懸浮液（suspension）和防腐劑組成的群組，其中上述組成物可以含有的物質常用於製備藥物。
醫藥組成物可以透過口服或非口服的方式施用。非口服的施用方式是由靜脈注射、皮下注射、肌內註射、腹膜內註射、內皮給藥、局部給藥、鼻腔給藥、肺內給藥、直腸給藥等。對於口服給藥，考慮到胜肽及蛋白質會在胃中分解，口服組合物中的活性成分需要配製成塗覆（coated）劑型或可以保護活性成分不致於在胃中分解的劑型。可替代地，本發明的組成物可以透過任何活性成分可以移動到目標細胞的裝置施用。
醫藥組成物可以配製為液體、懸浮液、糖漿或在油或水性介質中的乳劑（emulsion）的形式，或以萃取物、微粒、粉狀、顆粒、錠（tablet）或膠囊的形式，且為了配製目的可以額外包含分散劑或穩定劑。
尤其，根據本發明的用於治療癌症的組成物可以與其他抗癌藥物合併使用。
可以與根據本發明的新穎的PH20變異體或其片段合併使用的抗癌藥物較佳為化學抗癌藥物、基於抗體的抗癌藥物、生物抗癌藥物、RNAi或細胞治療劑，但不限於此。
較佳地，可與根據本發明的新穎的PH20變異體或其片段合併使用的抗癌藥物較佳為一免疫腫瘤試劑（immuno-oncologic agent），且更佳為一免疫檢查點抑制劑（immune checkpoint inhibitor），但不限於此。
此外，本發明涉及使用新穎的PH20變異體或其片段與其他抗癌藥物的組合以治療癌症的方法，尤其是上述的抗癌藥物。
另一方面，本發明涉及對PH20變異體或其片段進行編碼（encoding）的核酸。
如本文所用，核酸可以存在於細胞中、存在於細胞溶解產物（cell lysate）中或以部分純化或實質上純化的形式存在。當提及核酸時，「被分離」或「被實質上純化」是指已經透過標準技術純化並因此與其他細胞成分或其他污染物分離的核酸（例如其他核酸或蛋白質），所述標準技術包含鹼/SDS處理、CsCl顯帶（banding）、管柱層析法、瓊脂糖凝膠電泳（agarose gel electrophoresis）和其他本領域熟知的方法。本發明的核酸可以為DNA或RNA。
在又另一方面，本發明涉及重組表現載體（expression vector），包含核酸。對於根據本發明的PH20變異體或其片段的表現，DNA編碼PH20變異體或其片段可以透過標準分生技術取得（如聚合酶連鎖反應（polymerase chain reaction，PCR）放大技術或使用表現PH20變異體的融合瘤（hybridoma）的cDNA殖株（clone）），且DNA可以被插入表現載體使其與轉錄及轉譯控制序列「有效連接（operatively linked）」。
如本文所用，術語「有效連接」是旨在表示編碼PH20變異體或其片段的基因接合（ligated）到載體中，使得轉錄及轉譯控制序列起到調節編碼PH20變異體或其片段的基因的轉錄及轉譯的預期功能。所選的表現載體及表現控制序列為與所使用的表現宿主細胞相容。編碼PH20的基因透過標準方法（如連接編碼PH20變異體或其片段的基因片段上的互補限制性酶切位點（complementary restriction enzyme site）與載體，若不存在限制性酶切位點，則進行平滑端接合（blunt-end ligation））被插入表現載體。
此外，重組表現載體載有控制在宿主細胞中編碼PH20變異體或其片段的基因表現的調節序列（regulatory sequence）。所述「調節序列」旨在包含啟動子（promoter）、增強劑（enhancer）及其他控制編碼PH20變異體或其片段的基因的轉錄及轉譯的表現控制元素（如多腺苷酸化（polyadenylation）訊號）。本領域通常知識者將理解，表現載體的設計，包括調節序列的選擇，可取決於如以下的因素：待轉化的宿主細胞的選擇、所需的蛋白質表達程度等等。
在又一方面，本發明涉及宿主細胞，包含核酸或載體。根據本發明的宿主細胞較佳是從由動物細胞、植物細胞、酵母、大腸桿菌（E. coli）及昆蟲細胞組成的群組中所選擇，但不限於此。
具體地，根據本發明的宿主細胞包含原核細胞（prokaryotic cell）、菌類、酵母及真核細胞（eukaryotic cell）。原核細胞例如為大腸桿菌、枯草桿菌（Bacillus subtilis）、鏈黴菌屬（Streptomyces sp）、假單胞菌屬（Pseudomonas sp）、奇異變形桿菌（Proteus mirabilis）或葡萄球菌屬（Staphylococcus）。菌類例如為麴菌屬（Aspergillus sp）。酵母例如為嗜甲醇酵母菌（Pichia pastoris）、釀酒酵母（Saccharomyces cerevisiae）、裂殖酵母屬（Schizosaccharomyces sp）及紅麵包黴菌（Neurospora crass）。真核細胞例如為較低等的真核細胞，及其他較高等的真核細胞（如昆蟲細胞）。
此外，可用於本發明的宿主細胞可以從植物或哺乳類動物取得。較佳地，宿主細胞的例子包含但不限於：猴子腎細胞（COS7）、NSO cells、SP2/0、中國倉鼠卵（Chinese hamster ovary，CHO）細胞、W138、幼倉鼠腎（baby hamster kidney，BHK）細胞、MDCK、骨髓癌細胞（myeloma cell）、HuT 78細胞及HEK293細胞。更佳地，可以使用CHO細胞。
核酸或載體被轉染（transfect）進宿主細胞。轉染可以透過使用通常用於將外來的核酸（DNA或RNA）引入原核細胞或真核細胞的各種技術而被執行，例如，電泳、磷酸鈣沉澱（alcium phosphate precipitation）、DEAE-葡聚醣轉染（DEAE-dextran transfection）或脂質轉染（lipofection）。為了表現本發明的H20變異體或其片段重組表現載體與宿主細胞的各種組合可以被使用。用於真核細胞的較佳的表現載體包含基因表現調節序列、具有廣泛的宿主範圍的受體、以各種噬菌體λ衍生物為例（如λgt10、λgt11及NM989）的噬菌體DNA以及其他DNA噬菌體。基因表現調節序列是從SV40、胎牛乳突病毒（bovine papillomavirus）、腺病毒（adenovirus）、腺相關病毒（adeno-associated virus）、細胞巨大病毒（cytomegalovirus）及反轉錄病毒（retrovirus）取得，但不限於此。可以用於細菌宿主的表現載體包含從E. Coli取得的細菌受體，例如pET、pRSET、pBluescript、pGEX2T、pUC載體、col E1、pCR1、pBR322、pMB9及其衍生物。具有廣泛的宿主範圍的受體例如為RP4。所述其他DNA噬菌體例如為M13及絲狀單股DNA噬菌體（filamentous single-stranded DNA phage）。可用於酵母細胞的表現載體可以是2-μm受體及其衍生物。用於昆蟲細胞的表現載體包括pVL941。
在另一方面，本發明涉及用於製造PH20變異體或其片段的方法，該方法包含培養根據本發明的宿主細胞及表現PH20變異體或其片段。
當能夠表現PH20變異體或其片段的重組表現載體被引入哺乳動物的宿主細胞時，PH20變異體或其片段可以透過培養宿主細胞一段時間而被製造出來，使PH20變異體或其片段在宿主細胞中表現，其中所述一段時間較佳為使PH20變異體在宿主細胞的培養期間被分泌進培養液的一段時間。
在另一實施例中，可以表現的PH20變異體或其片段從宿主細胞中分離和純化出來。PH20變異體或其片段的分離和純化可以是以傳統用於蛋白質的分離/純化方法執行（如層析法）。層析法可以包含選自親和層析法（affinity chromatography）、離子交換層析法（ion exchange chromatography）及疏水作用層析法（hydrophobic chromatography）的一或多個組合，但不限於此。除了層析法，過濾、超濾、鹽析、透析等亦可以被使用。
為了確認酵素的產業利用性，有必要分析酵素的催化反應速率。酵素反應的類型包含具有固定反應性的活性部位（active site）的酵素反應及具有多種反應性的幾個活性部位的酵素反應。已知具有固定反應性的活性部位的酵素如玻尿酸酶的催化反應速率遵循Michaelis-Menten速率公式。
Figure 02_image001
Michaelis-Menten酵素動力學是建立在酵素反應為兩階段反應系統的假設上，所述兩階段反應系統包含形成酵素（E）－受質（S）的複合體［ES］的可逆反應階段及ES複合體解離以產生產物（P）的不可逆反應階段。在此情況下，kf 、kr 及kcat 為反應在每個方向上的速率常數（Alan Fersht（1977）酵素結構及機制）。
酵素反應假設與受質反應產生ES複合體的過程迅速達到平衡，或假設透過充分降低酵素的濃度而滿足d[ES]/dt≒0，則可以認為是擬穩態（pseudo-steady state），其中充分降低酵素的濃度是透過執行維持夠高的受質濃度實現。由於速率公式假設快速平衡及擬穩態是以相同方式推導得到，故在多數實驗中都假設了受質濃度最初高於酵素濃度的擬穩態。
Figure 02_image003
當「酵素量在反應前後為恆定」及「當化學反應達到化學平衡時，獲得產物的反應速率等於產物再次分解的速率」的情況作為假設時，最終產物的反應速率可以如下Michaelis-Menten速率公式表示。在此情況下，KM = (kr + kcat )/ kf ，且Vmax = kcat [E]0 。
Lineweaver-Burk方程式用於使用Michaelis-Menten速率公式以實驗性地分析酵素反應速率。此方程式示出了實驗中測得的反應速率的倒數1/V與實驗中給定受質濃度的倒數1/[S]之間的關係。此方程式為線性方程式的統計驗證展現了酵素反應為遵循Michaelis-Menten的速率公式的反應，且KM 及Vmax 可以透過使用此方程式而被計算出。
催化化學反應的酵素在於活性部位與受質結合後具有過渡態（transition state），且具有高能量的用於達到過渡態的活化能量透過與受質的多次結合而被降低。用於達成此過渡態的平衡常數與kcat /KM 成比例。於此，1/KM 為結合酵素—受質複合體透過將酵素結合至受質而產生的程度與酵素—受質複合體保持不變而不被分解的程度的指數，而kcat 為產物從酵素—受質複合體取得的平衡常數。因此，kcat /KM 可以被視為有多少產物能夠從受質與酵素取得的指標，即酵素的催化效率。
玻尿酸酶的產業利用性與其催化效率成比例。尤其，當該酵素與聚合藥理活性物質（polymeric pharmacologically active substance）一起注射至皮下時，玻尿酸酶的催化效率即扮演重要的腳色。在根據本發明的變異體具有較野生型PH20更高的kcat /KM 的情況下，當與聚合藥理活性物質合併的玻尿酸酶一起注射至皮下時，其中的玻尿酸被快速地分解並因此可獲得快速分散（disperse）聚合藥理活性物質的功效。此外，當根據本發明的變異體具有較野生型PH20更大的kcat 的情況下，最大反應速率Vmax 在相同酵素濃度下增加，進而在同樣時間內提供分解更大量的玻尿酸，及在更廣的區域分散聚合藥理活性物質的優異效果。
因此，為了證實根據本發明的PH20變異體的酵素特性，每個變異體的酵素反應速率皆被分析，且其Vmax （最大酵素反應速率）、KM （受質濃度為50%的Vmax 的情況）、kcat （受質轉換率）及kcat /KM （酵素催化效率）在例子4中被比較。上述的結果顯現出根據本發明的PH20變異體較野生型PH20更好。
例子
在下文中，將參考實施例更詳細地描述本發明。然而，對於本領域通常知識者顯而易見的是，提供這些實施例僅用於舉例說明本發明，而不應解釋為限制本發明的範圍。
例子1，建構PH20變異體
為建構PH20變異體，從韓國人類基因銀行（Korean Human Gene Bank）購買野生型PH20的cDNA（clone ID: hMU002604）。野生型PH20編碼從L36到S490的胺基酸。PH20基因透過聚合酶連鎖反應（polymerase chain reaction，以下簡稱PCR）被放大並被插入至pcDNA3.4-TOPO載體的限制酶部位XhoI及NotI。為ExpiCHO細胞中的表現，人類生長賀爾蒙、人類血清賀爾蒙或人類Hyal1的訊息肽被作為一訊息肽，而非PH20的原始訊息肽。為使用HisTrap管柱的蛋白質純化，His-tag的DNA序列位於PH20的cDNA的3’端。PH20變異體的胺基酸取代係使用PCR執行，且胺基酸取代係透過DNA測序確認。
用於選殖PH20變異體的引子（primer）列表已總結於如下表4，而特定的引子序列已總結於如下表5。
表4，用於選殖根據本發明的PH20變異體的引子的列表
殖株 引子
1 2 3
cB4205 ALB-SP-Xho B4-hy2 SPAM1-6H-not
cB4206 ALB-SP-Xho B4-hy3 SPAM1-6H-not
cB4207 ALB-SP-Xho B4-hy4 SPAM1-6H-not
cB4213-m63 opB4-Xho-hSA op-F468-6H-not 　-
cB4213-m64 opB4-Xho-hSA op-Q347-m64 op-F468-6H-not
cB4213-m65 op-Xho-hSA-L op-Q348-m65 op-F468-R
cB4213-m66 op-Xho-hSA-L op-Q350-m66 op-F468-R
cB4213-m67 opB4-Xho-hSA op-Q355-m67 op-F468-6H-not
cB4213-m69 op-Xho-hSA-L op-V358-m69 op-F468-6H-not
cB4213-m70 op-Xho-hSA-L op-A362-m70 op-F468-6H-not
cB4213-m71 opB4-Xho-hSA op-V343-m71 op-F468-6H-not
cB4213-m72 opB4-Xho-hSA op-F344-m72 op-F468-6H-not
cB4213-m73 op-Xho-hSA-L op-K359-mega-NL73 op-F468-6H-not
cB4213-m74 op-Xho-hSA-L op-Y360-m74 op-F468-6H-not
cB4213-m75 opB4-Xho-hSA op-M361-m75 op-F468-6H-not
cB4213-m76 opB4-Xho-hSA op-E352-m76 op-F468-6H-not
cB4213-m77 opB4-Xho-hSA op-M363-m77 op-F468-6H-not
cB4213-m78 opB4-Xho-hSA op-N84-m78 op-F468-6H-not
cB4213-m79 opB4-Xho-hSA op-K166-m79 op-F468-6H-not
cB4213-m82 op-Xho-hSA-L op-354E-m82 op-F468-6H-not
cB4213-m83 op-Xho-hSA-L op-354Q-m83 op-F468-6H-not
cB4213-m84 op-Xho-hSA-L op-354S-m84 op-F468-6H-not
cB4213-m85 op-Xho-hSA-L op-354V-m85 op-F468-6H-not
cB4213-m86 op-Xho-hSA-L op-354A-m86 op-F468-6H-not
cB4213-m88 op-Xho-hSA-L op-354N-m88 op-F468-6H-not
cB4213-m89 op-Xho-hSA-L op-354T-m89 op-F468-6H-not
cB4213-m90 op-Xho-hSA-L op-356M-m90 op-F468-6H-not
cB4213-m91 op-Xho-hSA-L op-356F-m91 op-F468-6H-not
cB4213-m92 op-Xho-hSA-L op-356I-m92 op-F468-6H-not
cB4213-m93 op-Xho-hSA-L op-356L-m93 op-F468-6H-not
cB4213-m94 op-Xho-hSA-L op-356Q-m94 op-F468-6H-not
cB4213-m95 op-Xho-hSA-L op-356V-m95 op-F468-6H-not
cB4213-m96 op-Xho-hSA-L op-343V_364M-m96 op-F468-6H-not
cB4213-m97 op-Xho-hSA-L op-340Q-m97 op-F468-6H-not
cB4213-m98 op-Xho-hSA-L op-341H-m98 op-F468-6H-not
cB4213-m99 op-Xho-hSA-L op-342I-m99 op-F468-6H-not
cB4213-m100 op-Xho-hSA-L op-343Y-m100 op-F468-6H-not
cB4213-m101 op-Xho-hSA-L op-345E-m101 op-F468-6H-not
cB4213-m102 op-Xho-hSA-L op-346F-m102 op-F468-6H-not
cB4213-m103 op-Xho-hSA-L op-347E-m103 op-F468-6H-not
cB4213-m104 op-Xho-hSA-L op-349L-m104 op-F468-6H-not
cB4213-m105 op-Xho-hSA-L op-350I-m105 op-F468-6H-not
cB4213-m106 op-Xho-hSA-L op-352G-m106 op-F468-6H-not
cB4213-m107 op-Xho-hSA-L op-354R-m107 op-F468-6H-not
cB4213-m110 op-Xho-hSA-L op-358R-m110 op-F468-6H-not
cB4213-m111 op-Xho-hSA-L op-359V-m111 op-F468-6H-not
cB4213-m112 op-Xho-hSA-L op-360R-m112 op-F468-6H-not
cB4213-m114 op-Xho-hSA-L op-345K-m114 op-F468-6H-not
cB4213-m115 op-Xho-hSA-L op-346L-m115 op-F468-6H-not
cB4213-m116 op-Xho-hSA-L op-347V-m116 op-F468-6H-not
cB4213-m117 op-Xho-hSA-L op-349W-m117 op-F468-6H-not
cB4213-m118 op-Xho-hSA-L op-354W-m118 op-F468-6H-not
cB4213-m121 op-Xho-hSA-L op-359Y-m121 op-F468-6H-not
cB4213-m125 op-Xho-hSA-L op-347W-m125 op-F468-6H-not
cB4213-m126 op-Xho-hSA-L op-357W-m126 op-F468-6H-not
cB4213-m130 op-Xho-hSA-L op-342D-m130 op-F468-6H-not
cB4213-m131 op-Xho-hSA-L op-343Q-m131 op-F468-6H-not
cB4213-m132 op-Xho-hSA-L op-347H-m132 op-F468-6H-not
cB4213-m133 op-Xho-hSA-L op-348F-m133 op-F468-6H-not
cB4213-m134 op-Xho-hSA-L op-350D-m134 op-F468-6H-not
cB4213-m135 op-Xho-hSA-L op-352Y-m135 op-F468-6H-not
cB4213-m136 op-Xho-hSA-L op-353E-m136 op-F468-6H-not
cB4213-m138 op-Xho-hSA-L op-358Y-m138 op-F468-6H-not
cB4213-m139 op-Xho-hSA-L op-359Q-m139 op-F468-6H-not
cB4213-m140 op-Xho-hSA-L op-360L-m140 op-F468-6H-not
cB4213-m141 op-Xho-hSA-L op-361E-m141 op-F468-6H-not
cB4213-m142 op-Xho-hSA-L op-363E-m142 op-F468-6H-not
cB4213-m143 op-Xho-hSA-L op-342H-m143 op-F468-6H-not
cB4213-m144 op-Xho-hSA-L op-348D-m144 op-F468-6H-not
cB4213-m145 op-Xho-hSA-L op-361H-m145 op-F468-6H-not
cB4213-m146 opB4-Xho-hSA Op-R39-m146-R op-F468-6H-not
cB4213-m147 opB4-Xho-hSA Op-A40-m147-R op-F468-6H-not
cB4213-m149 opB4-Xho-hSA op-D456-6H-not 　-
cB4213-m150 op-Xho-hSA-L op-350Q360R-m150 op-F468-6H-not
cB4213-m152 opB4-Xho-hSA Op-m152-D65A-R op-F468-6H-not
cB4213-m153 opB4-Xho-hSA Op-m153-E66A-R op-F468-6H-not
cB4213-m154 opB4-Xho-hSA Op-m154-P67A-R op-F468-6H-not
cB4213-m155 opB4-Xho-hSA Op-m155-L68A-R op-F468-6H-not
cB4213-m156 op-Xho-hSA-L op-311A-m156 op-F468-6H-not
cB4213-m157 op-Xho-hSA-L op-312A-m157 op-F468-6H-not
cB4213-m158 op-Xho-hSA-L op-313A-m158 op-F468-6H-not
cB4213-m159 op-Xho-hSA-L op-314A-m159 op-F468-6H-not
cB4213-m160 op-Xho-hSA-L N266A-m160 op-F468-6H-not
cB4213-m161 op-Xho-hSA-L T267A-m161 op-F468-6H-not
cB4213-m162 op-Xho-hSA-L Q268A-m162 op-F468-6H-not
cB4213-m163 op-Xho-hSA-L Q269A-m163 op-F468-6H-not
cB4213-m164 op-Xho-hSA-L P271A-m164 op-F468-6H-not
cB4213-m165 op-Xho-hSA-L V272A-m165 op-F468-6H-not
cB4213-m166 opB4-Xho-hSA Op-m166-I102A-R op-F468-6H-not
cB4213-m167 opB4-Xho-hSA Op-m167-D103A-R op-F468-6H-not
cB4213-m168 opB4-Xho-hSA Op-m168-S104A-R op-F468-6H-not
cB4213-m169 opB4-Xho-hSA Op-m169-I105A-R op-F468-6H-not
cB4213-m170 op-Xho-hSA-L op-m170-T132A-R op-F468-6H-not
cB4213-m171 op-Xho-hSA-L op-m171-F133A-R op-F468-6H-not
cB4213-m172 op-Xho-hSA-L op-m172-Y134A-R op-F468-6H-not
cB4213-m173 op-Xho-hSA-L V241A-m173 op-F468-6H-not
cB4213-m174 op-Xho-hSA-L E242A-m174 op-F468-6H-not
cB4213-m175 op-Xho-hSA-L I243A-m175 op-F468-6H-not
cB4213-m176 op-Xho-hSA-L K244A-m176 op-F468-6H-not
cB4213-m177 opB4-Xho-hSA Op-m177-L179A-R op-F468-6H-not
cB4213-m178 opB4-Xho-hSA Op-m178-S180A-R op-F468-6H-not
cB4213-m179 opB4-Xho-hSA Op-m179-L181A-R op-F468-6H-not
cB4213-m180 opB4-Xho-hSA Op-m180-T182A-R op-F468-6H-not
cB4213-m181 opB4-Xho-hSA Op-m181-T185A-R op-F468-6H-not
cB4213-m182 opB4-Xho-hSA Op-m182-E186A-R op-F468-6H-not
cB4213-m183 opB4-Xho-hSA Op-m183-K187A-R op-F468-6H-not
cB4213-m184 op-Xho-hSA-L op-K290A-m184 op-F468-6H-not
cB4213-m185 op-Xho-hSA-L op-I291A-m185 op-F468-6H-not
cB4213-m186 op-Xho-hSA-L op-P292A-m186 op-F468-6H-not
cB4213-m190 op-Xho-hSA-L L441A-m190 op-F468-6H-not
cB4213-m191 op-Xho-hSA-L S442A-m191 op-F468-6H-not
cB4213-m192 opB4-Xho-hSA op-D451A-m192 op-F468-6H-not
cB4213-m193 opB4-Xho-hSA op-T452A-m193 op-F468-6H-not
cB4213-m194 op-Xho-hSA-L op-D453A-m194 op-F468-6H-not
cB4213-m195 op-Xho-hSA-L op-D461A-6H-not op-F468-6H-not
cB4213-m196 op-Xho-hSA-L op-G462A-6H-not op-F468-6H-not
cB4213-m197 op-Xho-hSA-L op-V463A-6H-not op-F468-6H-not
cB4213-m198 op-Xho-hSA-L op-N82A-m198-R op-F468-6H-not
cB4213-m199 op-Xho-hSA-L op-N166A-m199-R op-F468-6H-not
cB4213-m203 op-Xho-hSA-L Op-S104N-m203-R op-F468-6H-not
cB4213-m204 op-Xho-hSA-L Op-I105Q-m204-R op-F468-6H-not
cB4213-m205 op-Xho-hSA-L op-Q268D-m205-F op-F468-6H-not
cB4213-m208 op-Xho-hSA-L op-Q268I-m208-F op-F468-6H-not
cB4213-m210 op-Xho-hSA-L op-291G-m210-F op-F468-6H-not
cB4213-m211 op-Xho-hSA-L op-292D-m211-F op-F468-6H-not
cB4213-m212 op-Xho-hSA-L op-T452D-m212 op-F468-6H-not
cB4213-m213 op-Xho-hSA-L op-T452H-m213 op-F468-6H-not
cB4213-m214 op-Xho-hSA-L op-T452K-m214 op-F468-6H-not
cB4213-m216 op-Xho-hSA-L Op-T452G-m216 op-F468-6H-not
cB4213-m217 op-Xho-hSA-L Op-T452P-m217 op-F468-6H-not
cB4213-m218 op-Xho-hSA-L op-T452M-m218 op-F468-6H-not
cB4213-m219 op-Xho-hSA-L op-T452F-m219 op-F468-6H-not
cB4213-m220 op-Xho-hSA-L op-D461R-6H-not-m220 op-F468-6H-not
cB4213-m231 op-Xho-hSA-L op-V463Y-6H-not-m231 　-
cB4213-m232 op-Xho-hSA-L op-S180T-R-m232 op-F468-6H-not
cB4213-m233 op-Xho-hSA-L op-D451S-F-m233 op-F468-6H-not
cB4213-m234 op-Xho-hSA-L op-L313P-m234-F op-F468-6H-not
cB4213-m235 op-Xho-hSA-L op-L313M-m235-F op-F468-6H-not
cB4213-m243 op-Xho-hSA-L op-L179S-m243-R op-F468-6H-not
cB4213-m245 op-Xho-hSA-L op-L179I-m245-R op-F468-6H-not
cB4213-m246 op-Xho-hSA-L op-L179F-m246-R op-F468-6H-not
cB4213-m254 op-Xho-hSA-L FQQ-Mega-m254 op-F468-6H-not
cB4213-m261 op-Xho-hSA-L op-Q268N-m259-m op-F468-6H-not
cB4213-m262 op-Xho-hSA-L B4-124-R op-F468-6H-not
cB4213-m263 op-Xho-hSA-L B4-124-R op-F468-6H-not
cB4213-m266 op-Xho-hSA-L op-L181M,E186D-m266 op-F468-6H-not
cB4213-m268 opB4-Xho-hSA op-Q268A-m268-m op-F468-6H-not
cB4213-m271 opB4-Xho-hSA op-344I,348M-m271 op-F468-6H-not
cB4213-m275 op-Xho-hSA-L op-DLSS-m275 op-F468-6H-not
cB4213-m276 op-Xho-hSA-L op-DLS-m276 op-F468-6H-not
cB4213-m279 opB4-Xho-hSA Op-K348M-m279 op-F468-6H-not
cB4213-m280 opB4-Xho-hSA Op-N344I K348M-m280 op-F468-6H-not
cB4213-m287 op-Xho-hSA-L Q268A-m162 op-F468-6H-not
cB4213-m288 opB4-Xho-hSA Q268A-m162 op-F468-6H-not
表5，用於選殖PH20變異體的引子序列
引子 SEQ ID NO 核苷酸（Nucleotide）序列(5' -＞ 3')
B4-hy2 7 ATA TGG GGA ACC CTC AGT ATA ACT ACA AGC ACT GAG ACC TGC CAA TAT CTG AAG GAT TAC CTG ACC AGA CTG CTG AAT CCT TAC ATA ATC AAC
B4-hy3 8 ATA TGG GGA ACC CTC AGT ATA TCC AGC AGT GAG GAA GAA TGC TGG CAT TTG CAC GAT TAC CTG GTA GAC ACA CTG AAT CCT TAC ATA ATC AAC
B4-hy4 9 ATA TGG GGA ACC CTC AGT ATA ACC GCA TCT AAG GCA AAC TGC ACA AAA GTA AAA CAA TTC GTC TCC AGT GAT CTG AAT CCT TAC ATA ATC AAC
ALB-SP-Xho 10 GAA TAT CTC GAG GCC ACC ATG AAG TGG GTT ACA
SPAM1-6H-not 11 CTA ATT GCG GCC GCT CAT TAG TGG TGA TGG TGA TGA TGG AAG AAA CCA ATT CTG C
op-F468-R 12 AAT TAG GCG GCC GCC TAT TAA AAG GCG TCG ATG CAC ACG CCA TC
op-F468-6H-not 13 CTC TAA TTG CGG CCG CTC ATT AGT GGT GAT GGT GAT GAT GAA AGG CGT CGA TGC ACA CGC CAT C
op-Xho-hSA-L 14 AAT TAG AGC TCG AGG CCA CCA TGA AAT GGG TGA CCT TTA TCT CC
opB4-Xho-hSA 15 CAG ATT CTC GAG GCC ACC ATG AAA TGG G
op-Q347-m64 16 ATC TGG GGC TCC TGG GAG AAC ACC AGG CAG AAG GAG AGC TGC CAG GCC ATC
op-Q348-m65 17  ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC CAG GAG AGC TGC CAG GCC ATC AAG
op-Q350-m66 18 AGA ACA CCA GGA CCA AGG AGC AAT GCC AGG CCA TCA AGG AGT AC
op-Q355-m67 19 AGG AGA GCT GCC AGG CCA TCC AGG AGT ACA TGG ACA CAA CCC TG
op-V358-m69 20 AGC TGC CAG GCC ATC AAG GAG TAC GTG GAC ACA ACC CTG AAC CCT TAT ATC
op-A362-m70 21 AGG AGT ACA TGG ACA CAA CCG CGA ACC CTT ATA TCA TCA ATG
op-V343-m71 22 ATC GTG ATC TGG GGC TCC TGG GTG AAC ACC AGG ACC AAG GAG AG
op-F344-m72 23 ATC TGG GGC TCC TGG GAG TTC ACC AGG ACC AAG GAG AGC TG
op-K359-mega-NL73 24 AGC TGC CAG GCC ATC AAG GAG TAC ATG AAA ACA ACC CTG AAC CCT TAT ATC
op-Y360-m74 25 ATC AAG GAG TAC ATG GAC TAC ACC CTG AAC CCT TAT ATC ATC
op-M361-m75 26 ATC AAG GAG TAC ATG GAC ACA ATG CTG AAC CCT TAT ATC ATC
op-E352-m76 27 ACC AGG ACC AAG GAG AGC TGC GAG GCC ATC AAG GAG TAC ATG G
op-M363-m77 28 AGT ACA TGG ACA CAA CCC TGA TGC CTT ATA TCA TCA ATG TGA C
op-N84-m78 29 TAG AAG ATT GTC ACG CCC TGG CCG TTG GCA TTG ATC CGA GGA GAG C
op-K166-m79 30 TGC ACC AGC TCG ATG GAC CGT TTC TTA TAC ACG TCC TTA GGC TTC
op-354E-m82 31 ACC AAG GAG AGC TGC CAG GCC GAA AAG GAG TAC ATG GAC ACA ACC
op-354Q-m83 32 ACC AAG GAG AGC TGC CAG GCC CAA AAG GAG TAC ATG GAC ACA ACC
op-354S-m84 33 ACC AAG GAG AGC TGC CAG GCC TCT AAG GAG TAC ATG GAC ACA ACC
op-354V-m85 34 ACC AAG GAG AGC TGC CAG GCC GTC AAG GAG TAC ATG GAC ACA ACC
op-354A-m86 35 ACC AAG GAG AGC TGC CAG GCC GCG AAG GAG TAC ATG GAC ACA ACC
op-354N-m88 36 ACC AAG GAG AGC TGC CAG GCC AAC AAG GAG TAC ATG GAC ACA ACC
op-354T-m89 37 ACC AAG GAG AGC TGC CAG GCC ACC AAG GAG TAC ATG GAC ACA ACC
op-356M-m90 38 AAG GAG AGC TGC CAG GCC ATC AAG ATG TAC ATG GAC ACA ACC CTG AAC
op-356F-m91 39 AAG GAG AGC TGC CAG GCC ATC AAG TTC TAC ATG GAC ACA ACC CTG AAC
op-356I-m92 40 AAG GAG AGC TGC CAG GCC ATC AAG ATA TAC ATG GAC ACA ACC CTG AAC
op-356L-m93 41 AAG GAG AGC TGC CAG GCC ATC AAG TTG TAC ATG GAC ACA ACC CTG AAC
op-356Q-m94 42 AAG GAG AGC TGC CAG GCC ATC AAG CAG TAC ATG GAC ACA ACC CTG AAC
op-356V-m95 43 AAG GAG AGC TGC CAG GCC ATC AAG GTA TAC ATG GAC ACA ACC CTG AAC
op-343V_364M-m96 44 ATC GTG ATC TGG GGC TCC TGG GTG AAC ACC AGG ACC AAG GAG AGC TGC CAG GCC ATC AAG GAG TAC ATG GAC ACA ATG CTG AAC CCT TAT ATC ATC
op-340Q-m97 45 AGC TAG CGG CAT CGT GAT CTG GCA ATC CTG GGA GAA CAC CAG GAC C
op-341H-m98 46 AGC GGC ATC GTG ATC TGG GGC CAC TGG GAG AAC ACC AGG ACC AAG
op-342I-m99 47 AGC GGC ATC GTG ATC TGG GGC TCC ATT GAG AAC ACC AGG ACC AAG GAG
op-343Y-m100 48 ATC GTG ATC TGG GGC TCC TGG TAT AAC ACC AGG ACC AAG GAG AG
op-345E-m101 49 ATC GTG ATC TGG GGC TCC TGG GAG AAC GAA AGG ACC AAG GAG AGC TGC C
op-346F-m102 50 ATC TGG GGC TCC TGG GAG AAC ACC TTC ACC AAG GAG AGC TGC CAG GC
op-347E-m103 51 ATC TGG GGC TCC TGG GAG AAC ACC AGG GAA AAG GAG AGC TGC CAG GCC ATC
op-349L-m104 52 ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC AAG TTG AGC TGC CAG GCC ATC AAG G
op-350I-m105 53 AGA ACA CCA GGA CCA AGG AGA TCT GCC AGG CCA TCA AGG AG
op-352G-m106 54 ACC AGG ACC AAG GAG AGC TGC GGG GCC ATC AAG GAG TAC ATG GAC
op-354R-m107 55 ACC AAG GAG AGC TGC CAG GCC AGA AAG GAG TAC ATG GAC ACA AC
op-358R-m110 56 AGC TGC CAG GCC ATC AAG GAG TAC CGG GAC ACA ACC CTG AAC CCT TAT ATC
op-359V-m111 57 AGG CCA TCA AGG AGT ACA TGG TCA CAA CCC TGA ACC CTT ATA TC
op-360R-m112 58 AGG CCA TCA AGG AGT ACA TGG ACA GAA CCC TGA ACC CTT ATA TCA TC
op-345K-m114 59 ATC TGG GGC TCC TGG GAG AAC AAG AGG ACC AAG GAG AGC TGC CAG
op-346L-m115 60 ATC TGG GGC TCC TGG GAG AAC ACC CTG ACC AAG GAG AGC TGC CAG GC
op-347V-m116 61 ATC TGG GGC TCC TGG GAG AAC ACC AGG GTC AAG GAG AGC TGC CAG GCC ATC
op-349W-m117 62 ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC AAG TGG AGC TGC CAG GCC ATC AAG GAG
op-354W-m118 63 ACC AAG GAG AGC TGC CAG GCC TGG AAG GAG TAC ATG GAC ACA AC
op-359Y-m121 64 AGG CCA TCA AGG AGT ACA TGT ACA CAA CCC TGA ACC CTT ATA TC
op-347W-m125 65 ATC TGG GGC TCC TGG GAG AAC ACC AGG TGG AAG GAG AGC TGC CAG GCC ATC
op-357W-m126 66 AGC TGC CAG GCC ATC AAG GAG TGG ATG GAC ACA ACC CTG AAC CC
op-342D-m130 67 AGC GGC ATC GTG ATC TGG GGC TCC GAC GAG AAC ACC AGG ACC AAG GAG
op-343Q-m131 68 ATC GTG ATC TGG GGC TCC TGG CAG AAC ACC AGG ACC AAG GAG AGC
op-347H-m132 69 ATC TGG GGC TCC TGG GAG AAC ACC AGG CAC AAG GAG AGC TGC CAG GCC ATC
op-348F-m133 70 ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC TTC GAG AGC TGC CAG GCC ATC AAG
op-350D-m134 71 AGA ACA CCA GGA CCA AGG AGG ACT GCC AGG CCA TCA AGG AGT AC
op-352Y-m135 72 ACC AGG ACC AAG GAG AGC TGC TAC GCC ATC AAG GAG TAC ATG GAC AC
op-353E-m136 73 AGG ACC AAG GAG AGC TGC CAG GAA ATC AAG GAG TAC ATG GAC AC
op-358Y-m138 74 AGC TGC CAG GCC ATC AAG GAG TAC TAC GAC ACA ACC CTG AAC CCT TAT ATC
op-359Q-m139 75 AGG CCA TCA AGG AGT ACA TGC AGA CAA CCC TGA ACC CTT ATA TC
op-360L-m140 76 AGG CCA TCA AGG AGT ACA TGG ACC TAA CCC TGA ACC CTT ATA TCA TC
op-361E-m141 77 ATC AAG GAG TAC ATG GAC ACA GAG CTG AAC CCT TAT ATC ATC AAT G
op-363E-m142 78 AGT ACA TGG ACA CAA CCC TGG AGC CTT ATA TCA TCA ATG TGA C
op-342H-m143 79 AGC GGC ATC GTG ATC TGG GGC TCC CAT GAG AAC ACC AGG ACC AAG GAG
op-348D-m144 80 ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC GAC GAG AGC TGC CAG GCC ATC AAG
op-361H-m145 81 ATC AAG GAG TAC ATG GAC ACA CAC CTG AAC CCT TAT ATC ATC AAT G
Op-R39-m146-R 82 TTT GGA ATC ACA GGA GGA GCC CGA GAG TAT GCG GAG CTA AAC AG
Op-A40-m147-R 83 TTT GGA ATC ACA GGA GGA GCA GAG TAT GCG GAG CTA AAC AG
op-D456-6H-not 84 CTC TAA TTG CGG CCG CCT ATT AGT GGT GAT GGT GAT GAT GGT CCA CGG CAT CTG TGT CCT TC
op-350Q360R-m150 85 AGA ACA CCA GGA CCA AGG AGC AGT GCC AGG CCA TCA AGG AGT ACA TGG ACC GAA CCC TGA ACC CTT ATA TCA TC
Op-m152-D65A-R 86 TAA AAG AGA ACA GGC TCA TAT CCA GGG GCT CGG CAA ACT TGC CCA GGC AGA ACT C
Op-m153-E66A-R 87 TAA AAG AGA ACA GGC TCA TAT CCA GGG GCG CGT CAA ACT TGC CCA GGC AGA AC
Op-m154-P67A-R 88 TAA AAG AGA ACA GGC TCA TAT CCA GGG CCT CGT CAA ACT TGC CCA GGC
Op-m155-L68A-R 89 TAA AAG AGA ACA GGC TCA TAT CCG CGG GCT CGT CAA ACT TGC CCA G
op-311A-m156 90 ACC AGG ATC GTG TTT ACA GAC GCG GTG CTG AAG TTC CTG TCC
op-312A-m157 91 AGG ATC GTG TTT ACA GAC CAG GCG CTG AAG TTC CTG TCC CAG
op-313A-m158 92 ATC GTG TTT ACA GAC CAG GTG GCG AAG TTC CTG TCC CAG GAT
op-314A-m159 93 ATC GTG TTT ACA GAC CAG GTG CTG GCG TTC CTG TCC CAG GAT GAG
N266A-m160 94 GCC CTG TAC CCT AGC ATC TAT CTG GCC ACC CAG CAG AGC CCA GTG GC
T267A-m161 95 CTG TAC CCT AGC ATC TAT CTG AAC GCC CAG CAG AGC CCA GTG GCC GCT AC
Q268A-m162 96 TAC CCT AGC ATC TAT CTG AAC ACC GCG CAG AGC CCA GTG GCC GCT ACA CTG
Q269A-m163 97 TAC CCT AGC ATC TAT CTG AAC ACC CAG GCG AGC CCA GTG GCC GCT ACA CTG TAT G
P271A-m164 98 AGC ATC TAT CTG AAC ACC CAG CAG AGC GCA GTG GCC GCT ACA CTG TAT GTG AGG
V272A-m165 99 TAT CTG AAC ACC CAG CAG AGC CCA GCG GCC GCT ACA CTG TAT GTG AGG
Op-m166-I102A-R 100 TGT CAC TCC GGT GAT AGA ATC GGC ATA TGG ATA GTA GCC CAG TCT G
Op-m167-D103A-R 101 TCA CTG TCA CTC CGG TGA TAG AAG CGA TAT ATG GAT AGT AGC CCA G
Op-m168-S104A-R 102 TCC GTT CAC TGT CAC TCC GGT GAT AGC ATC GAT ATA TGG ATA GTA GCC CAG
Op-m169-I105A-R 103 TCC GTT CAC TGT CAC TCC GGT GGC AGA ATC GAT ATA TGG ATA GTA GC
op-m170-T132A-R 104 TTG TCC ACT GGC ATG TAG AAG GCG ATG TCC TTC TTA GCC TTA TC
op-m171-F133A-R 105 TGC CCA GAT TGT CCA CTG GCA TGT AGG CGG TGA TGT CCT TCT TAG CCT TAT C
op-m172-Y134A-R 106 TGC CCA GAT TGT CCA CTG GCA TGG CGA AGG TGA TGT CCT TCT TAG
V241A-m173 107 AGA TCG TCG TTC CTC TTG ATC TCC GCA TTG AAA CAG GAG CCG TTG TAG CC
E242A-m174 108 GAC AGA TCG TCG TTC CTC TTG ATC GCC ACA TTG AAA CAG GAG CCG TTG TAG CC
I243A-m175 109 AGC CAA GAC AGA TCG TCG TTC CTC TTG GCC TCC ACA TTG AAA CAG GAG CCG TTG
K244A-m176 110 AGC CAA GAC AGA TCG TCG TTC CTC GCG ATC TCC ACA TTG AAA CAG GAG CCG
Op-m177-L179A-R 111 TCT GTG GCC TCG GTC AGG CTC GCC TGC ACG TTC TGC TGC TGC AC
Op-m178-S180A-R 112 TTC TCT GTG GCC TCG GTC AGG GCC AGC TGC ACG TTC TGC TGC TG
Op-m179-L181A-R 113 TAG CCT TCT CTG TGG CCT CGG TCG CGC TCA GCT GCA CGT TCT GCT G
Op-m180-T182A-R 114 TTA GCC TTC TCT GTG GCC TCG GCC AGG CTC AGC TGC ACG TTC TG
Op-m181-T185A-R 115 TCG AAC TCC TGC TTA GCC TTC TCT GCG GCC TCG GTC AGG CTC AGC TG
Op-m182-E186A-R 116 TCG AAC TCC TGC TTA GCC TTC GCT GTG GCC TCG GTC AGG CTC AG
Op-m183-K187A-R 117 TTC TCG AAC TCC TGC TTA GCC GCC TCT GTG GCC TCG GTC AGG C
op-K290A-m184 118 AGA GAG GCT ATC CGC GTG TCT GCG ATC CCC GAC GCC AAG TCC CCA C
op-I291A-m185 119 AGG CTA TCC GCG TGT CTA AGG CCC CCG ACG CCA AGT CCC CAC TG
op-P292A-m186 120 AGG CTA TCC GCG TGT CTA AGA TCG CCG ACG CCA AGT CCC CAC TGC CC
L441A-m190 121 AGT TTT ACT GCT CTT GTT ATT CCA CCG CGA GCT GTA AGG AGA AGG CTG ATG
S442A-m191 122 ACT GCT CTT GTT ATT CCA CCC TGG CCT GTA AGG AGA AGG CTG ATG TG
op-D451A-m192 123 AAG GAG AAG GCT GAT GTG AAG GCC ACA GAT GCC GTG GAC GTG TGC
op-T452A-m193 124 AAG GAG AAG GCT GAT GTG AAG GAC GCA GAT GCC GTG GAC GTG TGC ATC
op-D453A-m194 125 AAG GCT GAT GTG AAG GAC ACA GCT GCC GTG GAC GTG TGC ATC G
op-D461A-6H-not 126 ATA TTC GCG GCC GCC TAT TAG TGG TGA TGG TGA TGA TGA AAG GCG TCG ATG CAC ACG CCA GCA GCG ATG CAC ACG TCC ACG
op-G462A-6H-not 127 ATA TTC GCG GCC GCC TAT TAG TGG TGA TGG TGA TGA TGA AAG GCG TCG ATG CAC ACG GCA TCA GCG ATG CAC ACG TCC AC
op-V463A-6H-not 128 ATA TTC GCG GCC GCC TAT TAG TGG TGA TGG TGA TGA TGA AAG GCG TCG ATG CAC GCG CCA TCA GCG ATG CAC ACG
op-N82A-m198-R 129 TGT CAC GCC CTG GCC GGT GGC AGC GAT CCG AGG AGA GCC GAT AAA AG
op-N166A-m199-R 130 TGC ACC AGC TCG ATG GAC CGA GCC TTA TAC ACG TCC TTA GGC TTC
Op-S104N-m203-R 131 TTC ACT GTC ACT CCG GTG ATA TTA TCG ATA TAT GGA TAG TAG CC
Op-I105Q-m204-R 132 TCC GTT CAC TGT CAC TCC GGT CTG AGA ATC GAT ATA TGG ATA GTA GC
op-Q268D-m205-F 133 ACC CTA GCA TCT ATC TGA ACA CCG ATC AGA GCC CAG TGG CCG CTA C
op-Q268I-m208-F 134 ACC CTA GCA TCT ATC TGA ACA CCA TCC AGA GCC CAG TGG CCG CTA C
op-291G-m210-F 135 AGG CTA TCC GCG TGT CTA AGG GCC CCG ACG CCA AGT CCC CAC
op-292D-m211-F 136 ATC CGC GTG TCT AAG ATC GAC GAC GCC AAG TCC CCA CTG C
op-T452D-m212 137 AGA AGG CTG ATG TGA AGG ACG ACG ATG CCG TGG ACG TGT G
op-T452H-m213 138 AGA AGG CTG ATG TGA AGG ACC ACG ATG CCG TGG ACG TGT G
op-T452K-m214 139 AGA AGG CTG ATG TGA AGG ACA AAG ATG CCG TGG ACG TGT G
Op-T452G-m216 140 AGA AGG CTG ATG TGA AGG ACG GAG ATG CCG TGG ACG TGT G
Op-T452P-m217 141 AGA AGG CTG ATG TGA AGG ACC CAG ATG CCG TGG ACG TGT G
op-T452M-m218 142 AGA AGG CTG ATG TGA AGG ACA TGG ATG CCG TGG ACG TGT G
op-T452F-m219 143 AGA AGG CTG ATG TGA AGG ACT TCG ATG CCG TGG ACG TGT G
op-D461R-6H-not-m220 144 CTC TAA TTG CGG CCG CCT ATT AGT GGT GAT GGT GAT GAT GAA AGG CGT CGA TGC ACA CGC CCC TAG CGA TGC ACA CGT CCA C
op-V463Y-6H-not-m231 145 CTC TAA TTG CGG CCG CTC ATT AGT GGT GAT GGT GAT GAT GAA AGG CGT CGA TGC AGT AGC CAT CAG CGA TGC ACA C
op-S180T-R-m232 146 TTC TCT GTG GCC TCG GTC AGG GTC AGC TGC ACG TTC TGC TGC TG
op-D451S-F-m233 147 AGG AGA AGG CTG ATG TGA AGA GCA CAG ATG CCG TGG ACG TG
op-L313P-m234-F 148 ATC GTG TTT ACA GAC CAG GTG CCG AAG TTC CTG TCC CAG GAT GAG
op-L313M-m235-F 149 ATC GTG TTT ACA GAC CAG GTG ATG AAG TTC CTG TCC CAG GAT GAG
op-L179S-m243-R 150 TCT GTG GCC TCG GTC AGG CTC GAC TGC ACG TTC TGC TGC TGC AC
op-L179I-m245-R 151 TCT GTG GCC TCG GTC AGG CTA ATC TGC ACG TTC TGC TGC TGC AC
op-L179F-m246-R 152 TCT GTG GCC TCG GTC AGG CTA AAC TGC ACG TTC TGC TGC TGC AC
FQQ-Mega-m254 153 ATC GTG ATC TGG GGC TCC TGG GAG TTC ACC AGG ACC CAG GAG AGC TGC CAG GCC ATC CAG GAG TAC ATG GAC ACA ACC CTG AAC
op-Q268N-m259-m 154 ACC CTA GCA TCT ATC TGA ACA CCA ACC AGA GCC CAG TGG CCG CTA C
B4-124-R 155 GCC CAG GCA GAA CTC GC
op-L181M,E186D-m266 156 TCT CGA ACT CCT GCT TAG CCT TAT CTG TGG CCT CGG TCA TGC TCA GCT GCA CGT TCT GCT GC
op-Q268A-m268-m 157 ACC CTA GCA TCT ATC TGA ACA CCG CGC AGA GCC CAG TGG CCG CTA C
op-344I,348M-m271 158 ATC GTG ATC TGG GGC TCC TGG GAG ATC ACC AGG ACC ATG GAG AGC TGC CAG GCC ATC AAG
op-DLSS-m275 159 AGC GGC ATC GTG ATC TGG GGC GAC CTG TCG ATC TCC TCG ACC ATG GAG AGC TGC CAG GCC
op-DLS-m276 160 AGC GGC ATC GTG ATC TGG GGC GAC CTG TCG ATC TCC AGG ACC ATG GAG AGC TGC CAG
Op-K348M-m279 161 ATC TGG GGC TCC TGG GAG AAC ACC AGG ACC ATG GAG AGC TGC CAG GCC ATC AAG
Op-N344I K348M-m280 162 ATC GTG ATC TGG GGC TCC TGG GAG ATC ACC AGG ACC ATG GAG AGC TGC CAG GCC ATC AAG
在找到具有增加的酵素活性及熱穩定性的PH20變異體後，PH20變異體的不具有PH20變異體的cDNA亦被建構。
PH20變異體透過如下使用H20變異體的cDNA被建構。
變異體的表現是使用ExpiCHO表現系統所執行。當ExpiCHO細胞的細胞密度達6x106 /mL時，包含插入pcDNA3.4-TOPO載體的野生型PH20或PH20變異體cDNA的質體（plasmid）透過使用ExpiFectamine CHO反應劑（轉染試劑）而被轉染進ExpiCHO細胞。作為細胞培養基，ExpiCHO表現培養液（100到500mL）被使用。在轉染後，ExpiCHO細胞被在130 rpm震盪（shaking）下培養總共6天，在此期間將細胞在37°C下培養1天，然後在32°C的低溫下進一步培養5天。在完成培養後，細胞懸浮液透過以10000 rpm的轉速離心30分鐘的方式收集。
C端附有His-tag之野生型PH20及PH20變異體的重組蛋白（在ExpiCHO細胞中製造）透過使用AKTA引子系統（GE Healthcare Systems）的三個階段之管柱層析法來純化，且取決於變異體，所述三個階段的管柱層析法透過分別使用HisTrap HP管柱–Q Sepharose管柱–phenyl HP管柱及Q Sepharose管柱–HisTrap HP管柱–butyl HP管柱而被執行。
使用HisTrap HP管柱、Q Sepharose管柱及phenyl HP管柱的純化被以如下方式執行。對於使用HisTrap HP柱的蛋白質純化，緩衝液A（20 mM的磷酸鈉，pH 7.5，0.5 M的氯化鈉（NaCl））及緩衝液B（20 mM的磷酸鈉，pH 7.5，0.5 M的氯化鈉（NaCl），0.5 M的咪唑（imidazole））被製備。所述蛋白質與HisTrap HP管柱結合，且該管柱被以5管柱體積（column volume，CV）的緩衝液A沖洗以移除非特定結合蛋白。確認導電率（conductivity）維持在恆定的程度後，該管柱接著被以5 CV的20%緩衝液B沖洗以洗滌（elute）該蛋白。洗滌後的蛋白被以透析液（20 mM的磷酸鈉，pH 7.5，50 mM的氯化鈉）透析。對於使用Q Sepharose管柱的蛋白質純化，緩衝液A（20 mM的磷酸鈉，pH 7.5）及緩衝液B（20 mM的磷酸鈉，pH 7.5，0.5 M的氯化鈉）被製備。所述蛋白質與Q Sepharose管柱結合，且管該柱被以5 CV的緩衝液A沖洗以移除非特定結合蛋白，並接著以5 CV的緩衝液B以0到100%的濃度梯度沖洗以洗滌該蛋白。對於使用phenyl HP管柱的蛋白質純化，緩衝液A（20 mM的磷酸鈉，pH 7.0，1.5 M的硫酸銨（(NH4 )2 SO4 ））及緩衝液B（20 mM的磷酸鈉，pH 7.0）被製備。所述蛋白質與phenyl管柱結合，且該管柱被以5 CV的緩衝液A沖洗以移除非特定結合蛋白，並接著被以5 CV的緩衝液B以0到100%的濃度梯度沖洗以洗滌該蛋白。
使用Q Sepharose管柱、HisTrap HP管柱、butyl HP管柱的純化以如下方式執行。對於使用Q Sepharose柱的蛋白質純化，緩衝液A（20 mM的磷酸鈉（NaPi），15 mM的氯化鈉，pH 8.0）及緩衝液B（20 mM的磷酸鈉，500 mM的氯化鈉，pH 8.0）被製備。為了將培養液的pH值及傳導性調整為與緩衝液A相同，使用1 M Tris緩衝液（三羥甲基氨基甲烷緩衝液）將pH值滴定至8，並透過向其中添加水（PW）以將電導率調整至5 mS/cm或更小。接著，培養液透過具有0.22μm孔的膜被過濾。所述蛋白質與Q Sepharose管柱結合，且該管柱被以5 CV的緩衝液A沖洗以移除非特定結合蛋白，並接著以5 CV的緩衝液B沖洗以洗滌目標蛋白。對於使用HisTrap HP管柱的蛋白質純化，緩衝液A（20 mM的磷酸鈉，500 mM的氯化鈉，pH 7.5）及緩衝液B（20 mM的磷酸鈉，500 mM的氯化鈉，500 mM的咪唑，pH 7.5）被製備。所述蛋白質與HisTrap HP管柱結合，且該柱被以10 CV的7 %緩衝液B沖洗以移除非特定結合蛋白，並接著以3 CV的40 %緩衝液B沖洗以洗滌該蛋白。對於使用butyl HP管柱的蛋白質純化，緩衝液A（20 mM的磷酸鈉，1.5 M的硫酸銨（ammonium sulfate），pH 7.0）及緩衝液B（20 mM的磷酸鈉，pH 7.0）被製備。3 M的硫酸銨及欲裝載至該管柱上的蛋白樣品被以1:1的比例混和，且接著所產生的混合物透過具有0.22μm孔的膜被過濾。所述蛋白樣品結合至butyl HP管柱，且該管柱被以5 CV的緩衝液A沖洗以移除雜質。接著，目標蛋白被以0到100%的線性濃度梯度的緩衝液B洗滌，且被使用透析緩衝液（20 mM磷酸鈉，100 mM氯化鈉，pH 7.0）透析。根據本發明的變異體被以本發明所建議的方法純化，10 %的SDS-PAGE分析被執行於每個被純化的產物，且結果如圖1及3所示。
野生型PH20及PH20變異體的酵素活性係以濁度測試（turbidimetric assay）量測。
濁度測試為一種量測當玻尿酸與白蛋白（BSA）混合時產生的沉澱物中的吸光度的方法。當玻尿酸被PH20水解時，與白蛋白混和時產生的沉澱物的吸光度下降。濁度測試通常是以如下方式執行。玻尿酸酶PH20（Sigma）被稀釋至1、2、5、7.5、10、15、20、30、50及60 單位（units）/mL並製備於各試管中。純化的蛋白樣品被溶於酵素稀釋緩衝液（20 mM Tris．HCl（三羥甲基氨基甲烷鹽酸溶液），pH 7.0，77 mM氯化鈉，0.01% (w/v) 胎牛血清白蛋白），稀釋至100X、300X、600X、1200X及2400X，並製備於各別的試管。在在新的試管中，濃度3 mg/mL的玻尿酸溶液被稀釋10倍至0.3 mg/mL的濃度，以使每支試管中的體積變為180 μL。60 μL的酵素被加入至稀釋的玻尿酸溶液並與其及混合，並讓其在37℃下反應45分鐘。在反應完成後，50 μL的已反應酵素及250 μL的酸性白蛋白溶液被加入至96孔盤中的每個孔，並搖晃10分鐘，接著使用分光光譜儀（spectrophotometer）以600 nm量測其吸光度。
量測蛋白質的熱穩定度的方法包含透過動態光散射（dynamic light scattering，DLS）量測聚集溫度的方法、透過Sypro-Orange染劑在即時PCR（real-time PCR）中量測熔點溫度（Tm ）的方法，及在讓蛋白質在預定溫度下放置預定時間後量測酵素活性的方法等。在透過DLS量測聚集溫度的方法中，分子的聚集透過光散射被量測，並因此靈敏度高且聚集溫度通常是低於該蛋白質的熔點溫度。由於每個變異體被以製備為相同濃度（0.2 mg/mL）的溶液且接著被量測，故可以透過使用結果值作為聚集溫度來比較每個變異體的物理特性（Philo, J.S. (2009) Cur. Pharm. Biotech. 10, 359-372）。
本發明中藉由從具有SEQ ID NO: 3序列的PH20變異體的取代或切割胺基酸建構而成的PH20變異體的胺基酸序列如下表6所示。
在本發明中，實驗是進行在變異體上，其中所述變異體在表6所示的序列中的C端添加了用於蛋白質純化的六個組氨酸。此在C端的添加被發現並未影響酵素活性或蛋白質穩定度。根據本發明的變異體被命名為HM及序列號的組合，且根據例子3的變異體被命名為「Hyal2—變異體（Hyal2-variant）」、「Hyal3—變異體（Hyal3-variant）」及「Hyal4—變異體（Hyal4-variant）」。
表6，根據本發明的PH20變異體的胺基酸序列及其取代/切割特性
名稱 SEQ ID NO 取代 序列
Hyal2-variant 163 15 個胺基酸殘基S341T、W342L、E343S、N344I、R346T、T347S、K348T、S350T、A353Y、I354L、E356D、M358L、D359T及T360R、T361L從SEQ ID NO: 3被取代，切割發生在PH20的N端在胺基酸殘基 L36之前，且切割發生在PH20的C端的胺基酸殘基S490之後 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG TLSI T TST E T CQ YL K D Y LTR LLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS
Hyal3- variant 164 17個胺基酸殘基  S341T、W342L、E343S、N344I、T345S、R346S、T347S、K348E、S350E、Q352W、A353H、I354L、K355H、E356D、M358L、D359V及T360D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在胺基酸殘基之前，且切割發生在PH20的C端在胺基酸殘基 S490 之後 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG TLSISSSE E E C WHLHD Y LVD TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS
Hyal4- variant 165 17 胺基酸殘基  S341T、W342L、E343S、N344I、R346A、T347S、E349A、S350N、Q352T、A353K、I354V、E356Q、Y357F、M358V、D359S、T360S及T361D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在胺基酸殘基L36之前，且切割發生在PH20的C端在胺基酸殘基S490之後 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG TLSI T AS K AN C TKV K QFVSSD LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS
HM63 166 1個胺基酸殘基R346M從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468 胺基酸殘基 之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENT M TKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM64 167 1個胺基酸殘基 T347Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468 胺基酸殘基 之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTR Q KESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM65 168 1個胺基酸殘基 K348Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRT Q ESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM66 169 1個胺基酸殘基 S350Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKE Q CQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM67 170 1個胺基酸殘基 K355Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAI Q EYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM69 171 1個胺基酸殘基 M358V 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前及切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEY V DTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM70 172 1個胺基酸殘基 L362A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTT A NPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM71 173 1個胺基酸殘基 E343V 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSW V NTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM72 174 1個胺基酸殘基 N344F 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWE F TRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM73 175 1個胺基酸殘基 D359K 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYM K TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM74 176 1個胺基酸殘基 T360Y 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMD Y TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM75 177 1個胺基酸殘基 T361M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDT M LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM76 178 1個胺基酸殘基 Q352E 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESC E AIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM77 179 1個胺基酸殘基 N363M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTL M PYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM78 180 1個胺基酸殘基 T84N 從SEQ ID NO: 3被取代，殘基切割發生在PH20的N端在F38胺基酸之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINA N GQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM79 181 1個胺基酸殘基 N166K 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYK K RSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM82 182 1個胺基酸殘基 I354E 從SEQ ID NO: 3被取代，殘基切割發生在PH20的N端在F38胺基酸之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA E KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM83 183 1個胺基酸殘基 I354Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA Q KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM84 184 1個胺基酸殘基 I354S 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA S KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM85 185 1個胺基酸殘基 I354V 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA V KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM86 186 1個胺基酸殘基 I354A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA A KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM88 187 1個胺基酸殘基 I354N 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA N KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM89 188 1個胺基酸殘基 I354T 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA T KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM90 189 1個胺基酸殘基 E356M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIK M YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM91 190 1個胺基酸殘基 E356F 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIK F YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM92 191 1個胺基酸殘基 E356I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIK I YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM93 192 1個胺基酸殘基 E356L 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIK L YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM94 193 1個胺基酸殘基 E356Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIK Q YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM95 194 1個胺基酸殘基 E356V 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIK V YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM96 195 3 胺基酸殘基 N166K、E343V及T361M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYK K RSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSW V NTRTKESCQAIKEYMDT M LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM97 196 1個胺基酸殘基 G340Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIW Q SWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM98 197 1個胺基酸殘基 S341H 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG H WENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM99 198 1個胺基酸殘基 W342I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGS I ENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM100 199 1個胺基酸殘基 E343Y 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSW Y NTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM101 200 1個胺基酸殘基 T345E 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWEN E RTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM102 201 1個胺基酸殘基 R346F 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENT F TKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM103 202 1個胺基酸殘基 T347E 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTR E KESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM104 203 1個胺基酸殘基 E349L 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTK L SCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM105 204 1個胺基酸殘基 S350I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKE I CQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM106 205 1個胺基酸殘基 Q352G 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESC G AIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM107 206 1個胺基酸殘基 I354R 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA R KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM110 207 1個胺基酸殘基 M358R 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEY R DTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM111 208 1個胺基酸殘基 D359V 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYM V TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM112 209 1個胺基酸殘基 T360R 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMD R TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM114 210 1個胺基酸殘基 T345K 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWEN K RTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM115 211 1個胺基酸殘基 R346L 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENT L TKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM116 212 1個胺基酸殘基 T347V 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTR V KESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM117 213 1個胺基酸殘基 E349W 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTK W SCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM118 214 1個胺基酸殘基 I354W 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQA W KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM121 215 1個胺基酸殘基 D359Y 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前、及切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYM Y TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM125 216 1個胺基酸殘基 T347W 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTR W KESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM126 217 1個胺基酸殘基 Y357W 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKE W MDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM130 218 1個胺基酸殘基 W342D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGS D ENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM131 219 1個胺基酸殘基 E343Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSW Q NTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM132 220 1個胺基酸殘基 T347H 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTR H KESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM133 221 1個胺基酸殘基 K348F 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRT F ESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM134 222 1個胺基酸殘基 S350D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKE D CQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM135 223 1個胺基酸殘基 Q352Y 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESC Y AIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM136 224 1個胺基酸殘基 A353E 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQ E IKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM138 225 1個胺基酸殘基 M358Y 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEY Y DTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM139 226 1個胺基酸殘基 D359Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYM Q TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM140 227 1個胺基酸殘基 T360L 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMD L TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM141 228 1個胺基酸殘基 T361E 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDT E LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM142 229 1個胺基酸殘基 N363E 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTL E PYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM143 230 1個胺基酸殘基 W342H 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGS H ENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM144 231 1個胺基酸殘基 K348D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRT D ESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM145 232 1個胺基酸殘基 T361H 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDT H LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM146 233 無額外取代發生，切割發生在PH20的N端在R39在胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM147 234 無額外取代發生，切割發生在PH20的N端在A40胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 APPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM149 235 無額外取代發生，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在D456胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVD
HM150 236 2 胺基酸殘基 S350Q及T360R 從SEQ ID NO: 3被取代，切割發生在PH20的N端在R39胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKE Q CQAIKEYMD R TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM152 237 1個胺基酸殘基 D65A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKF A EPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM153 238 1個胺基酸殘基 E66A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFD A PLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM154 239 1個胺基酸殘基 P67A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDE A LDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM155 240 1個胺基酸殘基 L68A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEP A DMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM156 241 1個胺基酸殘基 Q311A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTD A VLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM157 242 1個胺基酸殘基 V312A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM158 243 1個胺基酸殘基 L313A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQV A KFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM159 244 1個胺基酸殘基 K314A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVL A FLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM160 245 1個胺基酸殘基 N266A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYL A TQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM161 246 1個胺基酸殘基 T267A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLN A QQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM162 247 1個胺基酸殘基 Q268A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT A QSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM163 248 1個胺基酸殘基 Q269A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQ A SPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM164 249 1個胺基酸殘基 P271A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQS A VAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM165 250 1個胺基酸殘基 V272A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSP A AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM166 251 1個胺基酸殘基 I102A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPY A DSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM167 252 1個胺基酸殘基 D103A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYI A SITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM168 253 1個胺基酸殘基 S104A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYID A ITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM169 254 1個胺基酸殘基 I105A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDS A TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM170 255 1個胺基酸殘基 T132A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI A FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM171 256 1個胺基酸殘基 F133A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDIT A YMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM172 257 1個胺基酸殘基 Y134A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITF A MPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM173 258 1個胺基酸殘基 V241A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN A EIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM174 259 1個胺基酸殘基 E242A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNV A IKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM175 260 1個胺基酸殘基 I243A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVE A KRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM176 261 1個胺基酸殘基 K244A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEI A RNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM177 262 1個胺基酸殘基 L179A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ A SLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM178 263 1個胺基酸殘基 S180A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQL A LTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM179 264 1個胺基酸殘基 L181A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM180 265 1個胺基酸殘基 T182A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSL A EATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM181 266 1個胺基酸殘基 T185A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEA A EKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM182 267 1個胺基酸殘基 E186A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEAT A KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM183 268 1個胺基酸殘基 K187A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATE A AKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM184 269 1個胺基酸殘基 K290A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVS A IPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM185 270 1個胺基酸殘基 I291A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSK A PDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM186 271 1個胺基酸殘基 P292A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKI A DAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM190 272 1個胺基酸殘基 L441A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYST A SCKEKADVKDTDAVDVCIADGVCIDAF
HM191 273 1個胺基酸殘基 S442A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL A CKEKADVKDTDAVDVCIADGVCIDAF
HM192 274 1個胺基酸殘基 D451A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVK A TDAVDVCIADGVCIDAF
HM193 275 1個胺基酸殘基 T452A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD A DAVDVCIADGVCIDAF
HM194 276 1個胺基酸殘基 D453A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDT A AVDVCIADGVCIDAF
HM195 277 1個胺基酸殘基 D461A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIA A GVCIDAF
HM196 278 1個胺基酸殘基 G462A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIAD A VCIDAF
HM197 279 1個胺基酸殘基 V463A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADG A CIDAF
HM198 280 1個胺基酸殘基 N82A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRI A ATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM199 281 1個胺基酸殘基 N166A 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYK A RSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM203 282 1個胺基酸殘基 S104N 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYID N ITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM204 283 1個胺基酸殘基 I105Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDS Q TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM205 284 1個胺基酸殘基 Q268D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT D QSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM208 285 1個胺基酸殘基 Q268I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT I QSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM210 286 1個胺基酸殘基 I291G 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSK G PDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM211 287 1個胺基酸殘基 P292D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKI D DAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM212 288 1個胺基酸殘基 T452D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM213 289 1個胺基酸殘基 T452H 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD H DAVDVCIADGVCIDAF
HM214 290 1個胺基酸殘基 T452K 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD K DAVDVCIADGVCIDAF
HM216 291 1個胺基酸殘基 T452G 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD G DAVDVCIADGVCIDAF
HM217 292 1個胺基酸殘基 T452P 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD P DAVDVCIADGVCIDAF
HM218 293 1個胺基酸殘基 T452M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD M DAVDVCIADGVCIDAF
HM219 294 1個胺基酸殘基 T452F 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD F DAVDVCIADGVCIDAF
HM220 295 1個胺基酸殘基 D461R 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIA R GVCIDAF
HM231 296 1個胺基酸殘基 V463Y 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADG Y CIDAF
HM232 297 1個胺基酸殘基 S180T 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQL T LTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM233 298 1個胺基酸殘基 D451S 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVK S TDAVDVCIADGVCIDAF
HM234 299 1個胺基酸殘基 L313P 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQV P KFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM235 300 1個胺基酸殘基 L313M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQV M KFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM243 301 1個胺基酸殘基 L179S 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ S SLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM245 302 1個胺基酸殘基 L179I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ I SLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM246 303 1個胺基酸殘基 L179F 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ F SLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM254 304 3 胺基酸殘基 N344F、K348Q及K355Q 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWE F TRT Q ESCQAI Q EYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM261 305 7 胺基酸殘基 T132S、L181A、E186D、Q268N、I291L、V312A、and T452D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI S FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEAT D KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT N QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM262 306 無額外取代發生，切割發生在PH20的N端在N37胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM263 307 無額外取代發生，切割發生在PH20的N端在L36胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM266 308 9 胺基酸殘基 R39K、I105A、T132S、L181M、E186D、I291L、Q268A、V312A及T452D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 F K APPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDS A TGVTVNGGIPQKISLQDHLDKAKKDI S FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS M TEAT D KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT A QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM268 309 7 胺基酸殘基 T132A、L181A、E186A、Q268A、I291L、V312A、and T452D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI A FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEAT A KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT A QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM271 310 2 胺基酸殘基 N344I及K348M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWE I TRT M ESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM275 311 10 胺基酸殘基 S341D、W342L、E343S、N344I、T345S、R346S、K348M、K355D、D359E及T361I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG DLSISS T M ESCQAI D EYM E T I LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM276 312 9 胺基酸殘基 S341D、W342L、E343S、N344I、T345S、K348M、K355D、D359E、and T361I 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWG DLSIS RT M ESCQAI D EYM E T I LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
HM279 313  8 胺基酸殘基 T132S、L181A、E186D、Q268N、I291L、V312A、T452D及K348M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI S FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEAT D KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT N QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWGSWENTRT M ESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM280 314  9 胺基酸殘基 T132S、L181A、E186D、Q268N、I291L、V312A、T452D、N344I、and K348M 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI S FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEAT D KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT N QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWGSWE I TRT M ESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM287 315  17 胺基酸殘基 T132A、L181A、E186A、Q268A、I291L、V312A、S341D、W342L、E343S、N344I、T345S、R346S、K348M、K355D、D359E、T361I、and T452D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI A FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEAT A KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT A QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWG DLSISS T M ESCQAI D EYM E T I LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
HM288 316 16 胺基酸殘基 T132A、L181A、E186A、Q268A、I291L、V312A、S341D、W342L、E343S、N344I、T345S、K348M、K355D、D359E、T361I及T452D 從SEQ ID NO: 3被取代，切割發生在PH20的N端在F38胺基酸殘基之前，且切割發生在PH20的C端在F468胺基酸殘基之後 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDI A FYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS A TEAT A KAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNT A QSPVAATLYVRNRVREAIRVSK L PDAKSPLPVFAYTRIVFTDQ A LKFLSQDELVYTFGETVALGASGIVIWG DLSIS RT M ESCQAI D EYM E T I LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKD D DAVDVCIADGVCIDAF
例子2，根據本發明的PH20變異體的特性
透過對變異體的研究對該蛋白質的結構和功能進行了進一步研究，其中變異體包含基於SEQ ID NO: 3之胺基酸序列在N端及C端的切割。表7所示的聚集溫度作為製備的變異體的表現量及活性的分析結果。
表現程度及特定活性係透過如例子1中所描述的濁度測試所分析。測試的結果如所示。此時，基於為培養液中純化後的各活性所設的定量極限（limit of quantification，LOQ），培養液中的活性超過300 unit/mL被標記為「＞LOQ」，而在純化後的活性超過15 unit/μg被標記為「＞LOQ」。在相反的情況中，不等式符號改變。活性分析的表現程度及定量極限及以此為基礎的測試結果如表7所示。SEQ ID NO: 1的野生型PH20（L36-Y482）的聚集溫度為46.5°C，而SEQ ID NO: 3的PH20變異體（F38-F468）的聚集溫度為51°C。
表7，根據本發明的PH20變異體的表現程度、特定活性及聚集溫度
變異體 從序列ID NO.3改變 表現程度（LOQ：300 units/mL） 特定活性（LOQ：15 units/μg） 聚集點 （℃）
胺基酸取代 始於 終於
HM63 R346M F38 F468 ＞ LOQ ＞ LOQ 52℃
HM64 T347Q F38 F468 ＞ LOQ ＞ LOQ 50℃
HM65 K348Q F38 F468 ＞ LOQ ＞ LOQ 51℃
HM66 S350Q F38 F468 ＞ LOQ ＞ LOQ 56℃
HM67 K355Q F38 F468 ＞ LOQ ＞ LOQ 50℃
HM69 M358V F38 F468 ＞ LOQ ＞ LOQ 50℃
HM70 L362A F38 F468 ＞ LOQ ＞ LOQ 48℃
HM71 E343V F38 F468 ＞ LOQ ＞ LOQ 50℃
HM72 N344F F38 F468 ＞ LOQ ＞ LOQ 52℃
HM73 D359K F38 F468 ＞ LOQ ＞ LOQ 50℃
HM74 T360Y F38 F468 ＞ LOQ ＞ LOQ 50℃
HM75 T361M F38 F468 ＞ LOQ ＞ LOQ 49℃
HM76 Q352E F38 F468 ＞ LOQ ＞ LOQ 52℃
HM77 N363M F38 F468 ＞ LOQ ＞ LOQ 58℃
HM78 T84N F38 F468 ＞ LOQ ＞ LOQ 48℃
HM79 N166K F38 F468 ＞ LOQ ＞ LOQ 49℃
HM82 I354E F38 F468 ＞ LOQ ＞ LOQ 49℃
HM83 I354Q F38 F468 ＞ LOQ ＞ LOQ 49℃
HM84 I354S F38 F468 ＞ LOQ ＞ LOQ 48℃
HM85 I354V F38 F468 ＞ LOQ ＞ LOQ 51℃
HM86 I354A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM88 I354N F38 F468 ＞ LOQ ＞ LOQ 49℃
HM89 I354T F38 F468 ＞ LOQ ＞ LOQ 49℃
HM90 E356M F38 F468 ＞ LOQ ＞ LOQ 50℃
HM91 E356F F38 F468 ＞ LOQ ＞ LOQ 49℃
HM92 E356I F38 F468 ＞ LOQ ＞ LOQ 49℃
HM93 E356L F38 F468 ＞ LOQ ＞ LOQ 49℃
HM94 E356Q F38 F468 ＞ LOQ ＞ LOQ 50℃
HM95 E356V F38 F468 ＞ LOQ ＞ LOQ 48℃
HM96 N166K, E343V, T361M F38 F468 ＞ LOQ ＞ LOQ 48℃
HM97 G340Q F38 F468 ＞ LOQ ＞ LOQ 50℃
HM98 S341H F38 F468 ＞ LOQ ＞ LOQ 51℃
HM99 W342I F38 F468 ＞ LOQ ＞ LOQ 51℃
HM100 E343Y F38 F468 ＞ LOQ ＞ LOQ 50℃
HM101 T345E F38 F468 ＞ LOQ ＞ LOQ 51℃
HM102 R346F F38 F468 ＞ LOQ ＞ LOQ 51℃
HM103 T347E F38 F468 ＞ LOQ ＞ LOQ 50℃
HM104 E349L F38 F468 ＞ LOQ ＞ LOQ 50℃
HM105 S350I F38 F468 ＞ LOQ ＞ LOQ 50℃
HM106 Q352G F38 F468 ＞ LOQ ＞ LOQ 49℃
HM107 I354R F38 F468 ＞ LOQ ＞ LOQ 48℃
HM110 M358R F38 F468 ＞ LOQ ＞ LOQ 48℃
HM111 D359V F38 F468 ＞ LOQ ＞ LOQ 50℃
HM112 T360R F38 F468 ＞ LOQ ＞ LOQ 52℃
HM114 T345K F38 F468 ＞ LOQ ＞ LOQ 50℃
HM115 R346L F38 F468 ＞ LOQ ＞ LOQ 52℃
HM116 T347V F38 F468 ＞ LOQ ＞ LOQ 49℃
HM117 E349W F38 F468 ＞ LOQ ＞ LOQ 49℃
HM118 I354W F38 F468 ＞ LOQ ＞ LOQ 51℃
HM121 D359Y F38 F468 ＞ LOQ ＞ LOQ 49℃
HM125 T347W F38 F468 ＞ LOQ ＞ LOQ 51℃
HM126 Y357W F38 F468 ＞ LOQ ＞ LOQ 55℃
HM130 W342D F38 F468 ＞ LOQ ＞ LOQ 51℃
HM131 E343Q F38 F468 ＞ LOQ ＞ LOQ 50℃
HM132 T347H F38 F468 ＞ LOQ ＞ LOQ 51℃
HM133 K348F F38 F468 ＞ LOQ ＞ LOQ 49℃
HM134 S350D F38 F468 ＞ LOQ ＞ LOQ 54℃
HM135 Q352Y F38 F468 ＞ LOQ ＞ LOQ 51℃
HM136 A353E F38 F468 ＞ LOQ ＞ LOQ 49℃
HM138 M358Y F38 F468 ＞ LOQ ＞ LOQ 48℃
HM139 D359Q F38 F468 ＞ LOQ ＞ LOQ 50℃
HM140 T360L F38 F468 ＞ LOQ ＞ LOQ 49℃
HM141 T361E F38 F468 ＞ LOQ ＞ LOQ 48℃
HM142 N363E F38 F468 ＞ LOQ ＞ LOQ 50℃
HM143 W342H F38 F468 ＞ LOQ ＞ LOQ 50℃
HM144 K348D F38 F468 ＞ LOQ ＞ LOQ 50℃
HM145 T361H F38 F468 ＞ LOQ ＞ LOQ 49℃
HM146 - R39 F468 ＞ LOQ ＞ LOQ 52℃
HM147 - A40 F468 ＞ LOQ ＞ LOQ 53℃
HM149 - F38 D456 ＞ LOQ ＞ LOQ 54℃
HM150 S350Q, T360R R39 F468 ＞ LOQ ＞ LOQ 50℃
HM152 D65A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM153 E66A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM154 P67A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM155 L68A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM156 Q311A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM157 V312A F38 F468 ＞ LOQ ＞ LOQ 56℃
HM158 L313A F38 F468 ＞ LOQ ＞ LOQ 55℃
HM159 K314A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM160 N266A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM161 T267A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM162 Q268A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM163 Q269A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM164 P271A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM165 V272A F38 F468 ＞ LOQ ＞ LOQ 52℃
HM166 I102A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM167 D103A F38 F468 ＞ LOQ ＞ LOQ 53℃
HM168 S104A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM169 I105A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM170 T132A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM171 F133A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM172 Y134A F38 F468 ＞ LOQ ＞ LOQ 52℃
HM173 V241A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM174 E242A F38 F468 ＜ LOQ ＞ LOQ 55℃
HM175 I243A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM176 K244A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM177 L179A F38 F468 ＞ LOQ ＞ LOQ 54℃
HM178 S180A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM179 L181A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM180 T182A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM181 T185A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM182 E186A F38 F468 ＞ LOQ ＞ LOQ 51℃
HM183 K187A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM184 K290A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM185 I291A F38 F468 ＞ LOQ ＞ LOQ 54℃
HM186 P292A F38 F468 ＞ LOQ ＞ LOQ 52℃
HM190 L441A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM191 S442A F38 F468 ＞ LOQ ＞ LOQ 54℃
HM192 D451A F38 F468 ＞ LOQ ＞ LOQ 54℃
HM193 T452A F38 F468 ＞ LOQ ＞ LOQ 53℃
HM194 D453A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM195 D461A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM196 G462A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM197 V463A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM198 N82A F38 F468 ＞ LOQ ＞ LOQ 49℃
HM199 N166A F38 F468 ＞ LOQ ＞ LOQ 50℃
HM203 S104N F38 F468 ＞ LOQ ＞ LOQ 48℃
HM204 I105Q F38 F468 ＞ LOQ ＞ LOQ 51℃
HM205 Q268D F38 F468 ＞ LOQ ＞ LOQ 55℃
HM208 Q268I F38 F468 ＜ LOQ ＞ LOQ 52℃
HM210 I291G F38 F468 ＜ LOQ ＞ LOQ 55℃
HM211 P292D F38 F468 ＜ LOQ ＞ LOQ 53℃
HM212 T452D F38 F468 ＞ LOQ ＞ LOQ 50℃
HM213 T452H F38 F468 ＞ LOQ ＞ LOQ 50℃
HM214 T452K F38 F468 ＞ LOQ ＞ LOQ 52℃
HM216 T452G F38 F468 ＞ LOQ ＞ LOQ 54℃
HM217 T452P F38 F468 ＞ LOQ ＞ LOQ 52℃
HM218 T452M F38 F468 ＞ LOQ ＞ LOQ 52℃
HM219 T452F F38 F468 ＞ LOQ ＞ LOQ 53℃
HM220 D461R F38 F468 ＞ LOQ ＞ LOQ 48℃
HM231 V463Y F38 F468 ＞ LOQ ＞ LOQ 51℃
HM232 S180T F38 F468 ＞ LOQ ＞ LOQ 51℃
HM233 D451S F38 F468 ＞ LOQ ＞ LOQ 51℃
HM234 L313P F38 F468 ＞ LOQ ＞ LOQ 49℃
HM235 L313M F38 F468 ＞ LOQ ＞ LOQ 52℃
HM243 L179S F38 F468 ＞ LOQ ＞ LOQ 52℃
HM245 L179I F38 F468 ＞ LOQ ＞ LOQ 48℃
HM246 L179F F38 F468 ＞ LOQ ＞ LOQ 55℃
HM254 N344F, K348Q, K355Q F38 F468 ＞ LOQ ＞ LOQ 53℃
HM261 T132S, L181A, E186D, Q268N, I291L, V312A, T452D F38 F468 ＞ LOQ ＞ LOQ 56℃
HM262 - N37 F468 ＞ LOQ ＞ LOQ 50℃
HM263 - L36 F468 ＞ LOQ ＞ LOQ 49℃
HM266 R39K, I105A, T132S, L181M, E186D, Q268A, I291L, V312A, T452D F38 F468 ＞ LOQ ＞ LOQ 57℃
HM268 T132A, L181A, E186A, Q268A, I291L, V312A, T452D F38 F468 ＞ LOQ ＞ LOQ 53℃
HM271 N344I,  K348M F38 F468 ＞ LOQ ＞ LOQ 51℃
HM275 S341D, W342L, E343S, N344I, T345S, R346S,  K348M,  K355D,  D359E,  T361I F38 F468 ＞ LOQ ＞ LOQ 48℃
HM276 S341D, W342L, E343S, N344I, T345S,  K348M,  K355D,  D359E,  T361I F38 F468 ＞ LOQ ＞ LOQ 48℃
HM279 T132S, L181A, E186D, Q268N, I291L, V312A, K348M, T452D F38 F468 ＞ LOQ ＞ LOQ 56℃
HM280 T132S, L181A, E186D, Q268N, I291L, V312A, N344I, K348M, T452D F38 F468 ＜ LOQ ＞ LOQ 59℃
HM287 T132A, L181A, E186A, Q268A, I291L, V312A, S341D, W342L, E343S, N344I, T345S, R346S,  K348M,  K355D,  D359E,  T361I, T452D F38 F468 ＜ LOQ ＞ LOQ 48℃
HM288 T132A, L181A, E186A, Q268A, I291L, V312A, S341D, W342L, E343S, N344I, T345S, K348M, K355D, D359E, T361I, T452D F38 F468 ＜ LOQ ＞ LOQ 48℃
從如上表7可知，在具有SEQ ID NO: 3之胺基酸序列的多個變異體中，總共有133種具有一個胺基酸殘基取代的變異體（即HM63、HM64、HM65、HM66、HM67、HM69、HM70、HM71、HM72、HM73、HM74、HM75、HM76、HM77、HM78、HM79、HM82、HM83、HM84、HM85、HM86、HM88、HM89、HM90、HM91、HM92、HM93、HM94、HM95、HM97、HM98、HM99、HM100、HM101、HM102、HM103、HM104、HM105、HM106、HM107、HM110、HM111、HM112、HM114、HM115、HM116、HM117、HM118、HM121、HM125、HM126、HM130、HM131、HM132、HM133、HM134、HM135、HM136、HM138、HM139、HM140、HM141、HM142、HM143、HM144、HM145、HM152、HM153、HM154、HM155、HM156、HM157、HM158、HM159、HM160、HM161、HM162、HM163、HM164、HM165、HM166、HM167、HM168、HM169、HM170、HM171、HM172、HM173、HM174、HM175、HM176、HM177、HM178、HM179、HM180、HM181、HM182、HM183、HM184、HM185、HM186、HM190、HM191、HM192、HM193、HM194、HM195、HM196、HM197、HM198、HM199、HM203、HM204、HM205、HM208、HM210、HM211、HM212、HM213、HM214、HM216、HM217、HM218、HM219、HM220、HM231、HM232、HM233、HM234、HM235、HM243、HM245及HM246）為在純化後在獲得的純化級分（purified fraction）中仍維持活性且具有48到58°C的聚集溫度的變異體，並因此展現良好的熱穩定性。其中，總共65種變異體（即HM63、HM64、HM65、HM66、HM67、HM69、HM70、HM71、HM72、HM73、HM74、HM75、HM76、HM77、HM78、HM79、HM82、HM83、HM84、HM85、HM86、HM88、HM89、HM90、HM91、HM92、HM93、HM94、HM95、HM98、HM99、HM100、HM101、HM102、HM103、HM104、HM105、HM106、HM107、HM110、HM111、HM112、HM114、HM115、HM116、HM117、HM118、HM121、HM125、HM126、HM130、HM131、HM132、HM133、HM134、HM135、HM136、HM138、HM139、HM140、HM141、HM142、HM143、HM144及HM145）為在從PH20的SEQ ID NO: 3的序列中多個取代部位的其中一個有所突變且具有48到58°C的聚集溫度的變異體。其中，總共68種變異體（HM97、HM152、HM153、HM154、HM155、HM156、HM157、HM158、HM159、HM160、HM161、HM162、HM163、HM164、HM165、HM166、HM167、HM168、HM169、HM170、HM171、HM172、HM173、HM174、HM175、HM176、HM177、HM178、HM179、HM180、HM181、HM182、HM183、HM184、HM185、HM186、HM190、HM191、HM192、HM193、HM194、HM195、HM196、HM197、HM198、HM199、HM203、HM204、HM205、HM208、HM210、HM211、HM212、HM213、HM214、HM216、HM217、HM218、HM219、HM220、HM231、HM232、HM233、HM234、HM235、HM243、HM245及HM246）為在從PH20的SEQ ID NO: 3中的取代部位之外的一個位置有所突變且具有48到56°C的聚集溫度的變異體。
因此，可以知道的是，不論取代位置為何，在從SEQ ID NO: 3的一個位置有取代的變異體相較於SEQ ID NO: 1的野生型PH20（L36-Y482）具有更高的聚集溫度。然而，在其中，HM174、HM208、HM210及HM211被發現在培養液中具有低於300 unit/mL（其為LOQ）的活性，但在純化後有高於15 unit/μg（其為LOQ）的活性。在此情況下，當僅在培養液中測定變異體的活性時，不能準確地分析變異體本身的特性。
此外，如以上表7所示，在具有SEQ ID NO: 3之胺基酸序列的變異體中，HM146、HM147、HM149、HM262及HM263保有與具有SEQ ID NO: 3之胺基酸序列的變異體相同的突變，所述突變即為胺基酸殘基的取代，但更包含在N端及C端的切割，表示具有SEQ ID NO: 3之胺基酸序列的變異體中的蛋白質的表現及活性不受進一步在N端及C端的切割影響。這些變異體具有49°C到53°C的聚集溫度，其與SEQ ID NO: 3的變異體的差異不大，表示變異體的物理特性亦不受進一步在N端及C端的切割影響。
此外，在具有SEQ ID NO: 3之胺基酸序列的變異體中，總共13種變異體（即HM96、HM150、HM254、HM261、HM266、HM268、HM271、HM275、HM276、HM279、HM280、HM287及HM288，其為在上表7列的變異體中包含一或多個胺基酸取代及切割的變異體）成功表現蛋白質，更保留了酵素活性，且具有48°C至59°C的聚集溫度。這表示即使在多個取代的情況中，仍能保留蛋白質的活性及物理特性。然而，多個取代表現出無法預測的酵素活性及聚集溫度，其中酵素活性及聚集溫度僅有在透過組成相同的每個單一取代中獲得的特徵組合才是無法預測的。
例子3，以序列Hyal2、Hyal3及Hyal4取代的變異體的活性分析
Hyal2（TTSTETCQYLKDYLTRL）、Hyal3（SSSEEECWHLHDYLVDT） 及Hyal4（TASKANCTKVKQFVSSD）的胺基酸序列是人體內除了Hyal1之外的玻尿酸酶的對應部分，取代了SEQ ID NO: 1的野生型PH20的胺基酸序列中M345至I361的位置，以研究蛋白質的穩定性如何變化。
藉由以對應序列Hyal2、Hyal3 及Hyal4取代成熟野生型PH20（L36-S490）的M345至I361的位置而建構成的變異體分別被稱為「Hyal2-變異體」、「Hyal3 -變異體」及「Hyal4-變異體」。
Hyal2-變異體、Hyal3 -變異體及Hyal4-變異體被建構，接著這些變異體的熱穩定性被分析（見圖2）。結果為，由DLS 量測得的Hyal2-變異體的聚集溫度為48°C，其較野生型PH20的46.5°C聚集溫度高出1.5°C，表示熱穩定性增加。
此外，為了確認這些變異體是否在ExpiCHO細胞培養中表現，這些變異體被以使用HisTrap管柱的同樣方式純化，且蛋白質的表現程度由SDS-PAGE分析進行比較。結果顯示Hyal3 -變異體的表現程度為最高，其次為Hyal2-變異體及Hyal4-變異體。
例子4，根據本發明的變異體的熱穩定性分析
SDS-PAGE分析被執行以確認根據本發明的變異體的熱穩定性。SEQ ID NO: 1（L36-Y482）的純化的野生型PH20及根據本發明的PH20變異體的SEQ ID NO: 3（F38-F468）的純化的蛋白質被儲存在42°C下連續7天，接著在降低及非降低狀態的情況下執行10%　SDS-PAGE分析（圖4）。
結果為，觀察到野生型PH20（L36-Y482）聚集（圖4中的行G），而SEQ ID NO: 3的PH20變異體（F38-F468）則未聚集（圖4中的行H）。發現這種聚集的差異是由於兩種蛋白質之間的聚集溫度差。據此，根據本發明的變異體被視為展現出更高的熱穩定性，並因其相較於野生型PH20有較高的聚集溫度而有望在產業上被廣泛地應用。
例子5，根據本發明的變異體的酵素動力學（enzyme kinetics）分析
為了分析根據本發明的變異體的酵素動力學，酵素活性透過Morgan-Elson方法被量測（Takahashi, T.等人（2003），Anal. Biochem. 322:257-263）。Morgan-Elson方法為比色法（colorimetric method），用於分析由玻尿酸酶與二甲基胺硼烷（para-dimethylaminobenzaldehyde ，DMAB）水解玻尿酸產生的乙醯基葡萄糖胺（N-acetyl-D-glucosamine，GlcNAc）的還原端反應生成的紅色物質（在545 nm），其為一種Ehrlich試劑。乙醯基葡萄糖胺（GlcNAc，Sigma）在稀釋緩衝液（0.1 M的磷酸鈉，0.1 M的氯化鈉，1.5 mM的葡萄糖二酸1,4-内酯（saccharic acid 1,4-lactone），pH 5.35）中被稀釋至0.25、0.50、0.75、1.00或1.25 mM，且在每個試管中被稀釋後的乙醯基葡萄糖胺被以四硼酸鹽（tetraborate）處理來還原，接著加入DMAB以誘發比色反應。在反應完後，以545 nm量測吸光度以產生GlcNAc的標準反應曲線。作為受質的玻尿酸在每個試管中被以稀釋緩衝液稀釋至0.54、0.65、0.87、1.23或2.17 μM，並在其中加入玻尿酸酶，接著讓其在37°C反應5分鐘並以100°C加熱5分鐘以終止酵素反應。酵素反應後得到的樣品透過用四硼酸鹽處理而還原，並加入DMAB以誘導比色反應。在反應完後，以545 nm量測吸光度，而酵素活性透過使用上述GlcNAc的標準反應曲線量測。SEQ ID NO: 1的野生型PH20及根據本發明的PH20變異體的酵素動力學透過使用此方法被分析。Lineweaver-Burk曲線的線性被偵測到以作為結果，表示根據本發明的PH20變異體遵循Michaelis-Menten酵素動力學方程式。
表8示出關於SEQ ID NO: 1的野生型PH20（L36-Y482）、SEQ ID NO: 3的PH20變異體（F38-F468）、HM261及HM268的酵素動力學分析結果獲得的Vmax （最大酵素反應速率）、KM （50%受質濃度）、kcat （受質轉換率）及kcat /KM （酵素催化效率）。可以看到的是，隨著KM 值降低，酵素的受質結合能力增加，而隨著kcat 值增加，酵素的受質轉換率增加，故每個PH20變異體的kcat /KM （酵素催化效率）高於野生型PH20的kcat /KM 。此外，SEQ ID NO: 3、HM261及HM268中的每一個的kcat 皆大於SEQ ID NO: 1的野生型PH20的kcat ，並因此酵素的受質轉換率大於SEQ ID NO: 1的野生型PH20的受質轉換率，故每個PH20變異體的產業可利用性皆大於野生型PH20。
表8，根據本發明之PH20變異體之酵素動力學分析之結果
Vmax (µM/sec) K M (µM) k cat (1/sec) k cat /K M
PH20 SEQ ID NO: 1 (L36-Y482) 4.5 ± 0.5 2.0 ± 0.3 30.6 ± 3.0 15.1 ± 1.1
SEQ ID NO: 3 (F38-F468) 3.7 ± 0.3 1.3 ± 0.0 47.6 ± 3.7 36.8 ± 2.0
HM261 5.2 ± 0.5 1.4 ± 0.2 33.9 ± 3.0 23.7 ± 2.2
HM268 2.9 ± 0.5 0.9 ± 0.2 37.1 ± 6.4 40.0 ± 3.4
無
從以下結合附圖的詳細描述中，將更清楚地理解本發明的上述和其他目的、特徵和其他優點，其中： 圖1示出基於具有SEQ ID NO: 3之胺基酸序列的PH20變異體的各種變異體所執行之十二烷基硫酸鈉聚丙烯酰胺凝膠電泳（SDS-PAGE）分析的結果，以下關於各變異體的SDS-PAGE分析結果是透過管柱層析法（column chromatography）純化表達每個變異體的動物細胞培養液，並對最終純化的變異體執行10%的SDS-PAGE分析而取得； 圖1A更具體地示出關於變異體HM98、HM99、HM130、HM143、HM71、HM100、HM131、HM72、HM101及HM114的SDS-PAGE的結果； 圖1B示出關於變異體HM63、HM102、HM115、HM64、HM103、HM116、HM125、HM132、HM65、HM133、HM144、HM104及HM117的SDS-PAGE的結果； 圖1C示出關於變異體HM66、HM105、HM134、HM76、HM106、HM135、HM136及HM67的SDS-PAGE膠的結果； 圖1D示出關於變異體HM82、HM83、HM84、HM85、HM86、HM88、HM89、HM107、HM118、HM90、HM91、HM92、HM93、HM94及HM95的SDS-PAGE的結果； 圖1E示出關於變異體HM73、HM111、HM121、HM139、HM74、HM112及HM140的SDS-PAGE的結果； 圖1F示出關於變異體HM75、HM141、HM145、HM70、HM77、HM142、HM78、HM79、HM96、HM146、HM147、HM149及HM150的SDS-PAGE的結果； 圖2示出成熟的野生型PH20、Hyal2變異體、Hyal3變異體及Hyal4變異體的表現程度，其中Hyal2變異體、Hyal3變異體及Hyal4變異體為野生型PH20的區域M345到I361分別被替換為Hyal2、Hyal3及Hyal4的對應序列，其中SDS-PAGE的行CS為培養液樣本，SDS-PAGE的行FT為組氨酸標籤（HisTag）柱的未結合的雜質，而行E為HisTag柱的析出液； 圖3示出基於具有SEQ ID NO: 3之胺基酸序列的PH20變異體，對各種變異體執行SDS-PAGE分析的結果，以下關於各變異體的SDS-PAGE分析結果是透過管柱層析法純化表達每個變異體的動物細胞培養液，並對最終純化的變異體執行10%的SDS-PAGE分析而取得； 圖3A示出關於變異體HM152、HM153、HM154、HM155、HM156、HM157、HM158、HM159、HM160、HM161、HM162、HM163、HM164、HM165、HM166、HM167、HM168及HM169的SDS-PAGE的結果； 圖3B示出關於變異體HM170、HM171、HM172、HM173、HM174、HM175、HM176、HM177、HM178、HM179、HM180、HM181、HM182、HM183、HM184、HM185及HM186的SDS-PAGE的結果； 圖3C示出關於變異體HM190、HM191、HM192、HM193、HM194、HM195、HM196、HM197、HM198、HM199、HM203、HM204及HM205的SDS-PAGE的結果； 圖3D示出關於變異體HM208、HM210、HM211、HM212、HM213、HM214、HM216、HM217、HM218、HM219及HM220的SDS-PAGE的結果； 圖3E示出關於變異體HM231、HM232、HM233、HM234、HM235、HM243、HM245及HM246的SDS-PAGE的結果； 圖3F示出關於變異體HM254、HM261、HM262、HM263、HM266、HM268、HM271、HM275、HM276、HM279、HM280、HM287及HM288的SDS-PAGE的結果；以及 圖4示出SDS-PAGE的結果，確認了野生型PH20（L36-Y482）及具有SEQ ID NO: 3之胺基酸序列的PH20變異體（F38-F468）的熱穩定性，其中行A、B、C及D示出以下的SDS-PAGE分析的結果：關於初始的野生型PH20（行A及C）及在還原型（reduced form）（行A及B）以及非還原型（行C及D）中的SEQ ID NO: 3的PH20變異體（行B及D），而行E、F、G及H示出了以下各樣本在42°C下被儲存七天後的SDS-PAGE分析的結果：關於初始的野生型PH20（行E及G）及在還原型（行E及F）以及非還原型（行G及H）中的SEQ ID NO: 3的PH20變異體（行F及H）。
Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0026
Figure 12_A0101_SEQ_0027
Figure 12_A0101_SEQ_0028
Figure 12_A0101_SEQ_0029
Figure 12_A0101_SEQ_0030
Figure 12_A0101_SEQ_0031
Figure 12_A0101_SEQ_0032
Figure 12_A0101_SEQ_0033
Figure 12_A0101_SEQ_0034
Figure 12_A0101_SEQ_0035
Figure 12_A0101_SEQ_0036
Figure 12_A0101_SEQ_0037
Figure 12_A0101_SEQ_0038
Figure 12_A0101_SEQ_0039
Figure 12_A0101_SEQ_0040
Figure 12_A0101_SEQ_0041
Figure 12_A0101_SEQ_0042
Figure 12_A0101_SEQ_0043
Figure 12_A0101_SEQ_0044
Figure 12_A0101_SEQ_0045
Figure 12_A0101_SEQ_0046
Figure 12_A0101_SEQ_0047
Figure 12_A0101_SEQ_0048
Figure 12_A0101_SEQ_0049
Figure 12_A0101_SEQ_0050
Figure 12_A0101_SEQ_0051
Figure 12_A0101_SEQ_0052
Figure 12_A0101_SEQ_0053
Figure 12_A0101_SEQ_0054
Figure 12_A0101_SEQ_0055
Figure 12_A0101_SEQ_0056
Figure 12_A0101_SEQ_0057
Figure 12_A0101_SEQ_0058
Figure 12_A0101_SEQ_0059
Figure 12_A0101_SEQ_0060
Figure 12_A0101_SEQ_0061
Figure 12_A0101_SEQ_0062
Figure 12_A0101_SEQ_0063
Figure 12_A0101_SEQ_0064
Figure 12_A0101_SEQ_0065
Figure 12_A0101_SEQ_0066
Figure 12_A0101_SEQ_0067
Figure 12_A0101_SEQ_0068
Figure 12_A0101_SEQ_0069
Figure 12_A0101_SEQ_0070
Figure 12_A0101_SEQ_0071
Figure 12_A0101_SEQ_0072
Figure 12_A0101_SEQ_0073
Figure 12_A0101_SEQ_0074
Figure 12_A0101_SEQ_0075
Figure 12_A0101_SEQ_0076
Figure 12_A0101_SEQ_0077
Figure 12_A0101_SEQ_0078
Figure 12_A0101_SEQ_0079
Figure 12_A0101_SEQ_0080
Figure 12_A0101_SEQ_0081
Figure 12_A0101_SEQ_0082
Figure 12_A0101_SEQ_0083
Figure 12_A0101_SEQ_0084
Figure 12_A0101_SEQ_0085
Figure 12_A0101_SEQ_0086
Figure 12_A0101_SEQ_0087
Figure 12_A0101_SEQ_0088
Figure 12_A0101_SEQ_0089
Figure 12_A0101_SEQ_0090
Figure 12_A0101_SEQ_0091
Figure 12_A0101_SEQ_0092
Figure 12_A0101_SEQ_0093
Figure 12_A0101_SEQ_0094
Figure 12_A0101_SEQ_0095
Figure 12_A0101_SEQ_0096
Figure 12_A0101_SEQ_0097
Figure 12_A0101_SEQ_0098
Figure 12_A0101_SEQ_0099
Figure 12_A0101_SEQ_0100
Figure 12_A0101_SEQ_0101
Figure 12_A0101_SEQ_0102
Figure 12_A0101_SEQ_0103
Figure 12_A0101_SEQ_0104
Figure 12_A0101_SEQ_0105
Figure 12_A0101_SEQ_0106
Figure 12_A0101_SEQ_0107
Figure 12_A0101_SEQ_0108
Figure 12_A0101_SEQ_0109
Figure 12_A0101_SEQ_0110
Figure 12_A0101_SEQ_0111
Figure 12_A0101_SEQ_0112
Figure 12_A0101_SEQ_0113
Figure 12_A0101_SEQ_0114
Figure 12_A0101_SEQ_0115
Figure 12_A0101_SEQ_0116
Figure 12_A0101_SEQ_0117
Figure 12_A0101_SEQ_0118
Figure 12_A0101_SEQ_0119
Figure 12_A0101_SEQ_0120
Figure 12_A0101_SEQ_0121
Figure 12_A0101_SEQ_0122
Figure 12_A0101_SEQ_0123
Figure 12_A0101_SEQ_0124
Figure 12_A0101_SEQ_0125
Figure 12_A0101_SEQ_0126
Figure 12_A0101_SEQ_0127
Figure 12_A0101_SEQ_0128
Figure 12_A0101_SEQ_0129
Figure 12_A0101_SEQ_0130
Figure 12_A0101_SEQ_0131
Figure 12_A0101_SEQ_0132
Figure 12_A0101_SEQ_0133
Figure 12_A0101_SEQ_0134
Figure 12_A0101_SEQ_0135
Figure 12_A0101_SEQ_0136
Figure 12_A0101_SEQ_0137
Figure 12_A0101_SEQ_0138
Figure 12_A0101_SEQ_0139
Figure 12_A0101_SEQ_0140
Figure 12_A0101_SEQ_0141
Figure 12_A0101_SEQ_0142
Figure 12_A0101_SEQ_0143
Figure 12_A0101_SEQ_0144
Figure 12_A0101_SEQ_0145
Figure 12_A0101_SEQ_0146
Figure 12_A0101_SEQ_0147
Figure 12_A0101_SEQ_0148
Figure 12_A0101_SEQ_0149
Figure 12_A0101_SEQ_0150
Figure 12_A0101_SEQ_0151
Figure 12_A0101_SEQ_0152
Figure 12_A0101_SEQ_0153
Figure 12_A0101_SEQ_0154
Figure 12_A0101_SEQ_0155
Figure 12_A0101_SEQ_0156
Figure 12_A0101_SEQ_0157
Figure 12_A0101_SEQ_0158
Figure 12_A0101_SEQ_0159
Figure 12_A0101_SEQ_0160
Figure 12_A0101_SEQ_0161
Figure 12_A0101_SEQ_0162
Figure 12_A0101_SEQ_0163
Figure 12_A0101_SEQ_0164
Figure 12_A0101_SEQ_0165
Figure 12_A0101_SEQ_0166
Figure 12_A0101_SEQ_0167
Figure 12_A0101_SEQ_0168
Figure 12_A0101_SEQ_0169
Figure 12_A0101_SEQ_0170
Figure 12_A0101_SEQ_0171
Figure 12_A0101_SEQ_0172
Figure 12_A0101_SEQ_0173
Figure 12_A0101_SEQ_0174
Figure 12_A0101_SEQ_0175
Figure 12_A0101_SEQ_0176
Figure 12_A0101_SEQ_0177
Figure 12_A0101_SEQ_0178
Figure 12_A0101_SEQ_0179
Figure 12_A0101_SEQ_0180
Figure 12_A0101_SEQ_0181
Figure 12_A0101_SEQ_0182
Figure 12_A0101_SEQ_0183
Figure 12_A0101_SEQ_0184
Figure 12_A0101_SEQ_0185
Figure 12_A0101_SEQ_0186
Figure 12_A0101_SEQ_0187
Figure 12_A0101_SEQ_0188
Figure 12_A0101_SEQ_0189
Figure 12_A0101_SEQ_0190
Figure 12_A0101_SEQ_0191
Figure 12_A0101_SEQ_0192
Figure 12_A0101_SEQ_0193
Figure 12_A0101_SEQ_0194
Figure 12_A0101_SEQ_0195
Figure 12_A0101_SEQ_0196
Figure 12_A0101_SEQ_0197
Figure 12_A0101_SEQ_0198
Figure 12_A0101_SEQ_0199
Figure 12_A0101_SEQ_0200
Figure 12_A0101_SEQ_0201
Figure 12_A0101_SEQ_0202
Figure 12_A0101_SEQ_0203
Figure 12_A0101_SEQ_0204
Figure 12_A0101_SEQ_0205
Figure 12_A0101_SEQ_0206
Figure 12_A0101_SEQ_0207
Figure 12_A0101_SEQ_0208
Figure 12_A0101_SEQ_0209
Figure 12_A0101_SEQ_0210
Figure 12_A0101_SEQ_0211
Figure 12_A0101_SEQ_0212
Figure 12_A0101_SEQ_0213
Figure 12_A0101_SEQ_0214
Figure 12_A0101_SEQ_0215
Figure 12_A0101_SEQ_0216
Figure 12_A0101_SEQ_0217
Figure 12_A0101_SEQ_0218
Figure 12_A0101_SEQ_0219
Figure 12_A0101_SEQ_0220
Figure 12_A0101_SEQ_0221
Figure 12_A0101_SEQ_0222
Figure 12_A0101_SEQ_0223
Figure 12_A0101_SEQ_0224
Figure 12_A0101_SEQ_0225
Figure 12_A0101_SEQ_0226
Figure 12_A0101_SEQ_0227
Figure 12_A0101_SEQ_0228
Figure 12_A0101_SEQ_0229
Figure 12_A0101_SEQ_0230
Figure 12_A0101_SEQ_0231
Figure 12_A0101_SEQ_0232
Figure 12_A0101_SEQ_0233
Figure 12_A0101_SEQ_0234
Figure 12_A0101_SEQ_0235
Figure 12_A0101_SEQ_0236
Figure 12_A0101_SEQ_0237
Figure 12_A0101_SEQ_0238
Figure 12_A0101_SEQ_0239
Figure 12_A0101_SEQ_0240
Figure 12_A0101_SEQ_0241
Figure 12_A0101_SEQ_0242
Figure 12_A0101_SEQ_0243
Figure 12_A0101_SEQ_0244
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Figure 12_A0101_SEQ_0246
Figure 12_A0101_SEQ_0247
Figure 12_A0101_SEQ_0248
Figure 12_A0101_SEQ_0249
Figure 12_A0101_SEQ_0250
Figure 12_A0101_SEQ_0251
Figure 12_A0101_SEQ_0252
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Figure 12_A0101_SEQ_0255
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Figure 12_A0101_SEQ_0257
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Figure 12_A0101_SEQ_0260
Figure 12_A0101_SEQ_0261
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Figure 12_A0101_SEQ_0270
Figure 12_A0101_SEQ_0271
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Figure 12_A0101_SEQ_0281
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Figure 12_A0101_SEQ_0289
Figure 12_A0101_SEQ_0290
Figure 12_A0101_SEQ_0291
Figure 12_A0101_SEQ_0292
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Figure 12_A0101_SEQ_0294
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Figure 12_A0101_SEQ_0296
Figure 12_A0101_SEQ_0297
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Figure 12_A0101_SEQ_0299
Figure 12_A0101_SEQ_0300
Figure 12_A0101_SEQ_0301
Figure 12_A0101_SEQ_0302
Figure 12_A0101_SEQ_0303
Figure 12_A0101_SEQ_0304
Figure 12_A0101_SEQ_0305
Figure 12_A0101_SEQ_0306
Figure 12_A0101_SEQ_0307
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Figure 12_A0101_SEQ_0310
Figure 12_A0101_SEQ_0311
Figure 12_A0101_SEQ_0312
Figure 12_A0101_SEQ_0313
Figure 12_A0101_SEQ_0314
Figure 12_A0101_SEQ_0315
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Figure 12_A0101_SEQ_0318
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Figure 12_A0101_SEQ_0320
Figure 12_A0101_SEQ_0321
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Figure 12_A0101_SEQ_0323
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Figure 12_A0101_SEQ_0325
Figure 12_A0101_SEQ_0326
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Figure 12_A0101_SEQ_0330
Figure 12_A0101_SEQ_0331
Figure 12_A0101_SEQ_0332
Figure 12_A0101_SEQ_0333
Figure 12_A0101_SEQ_0334
Figure 12_A0101_SEQ_0335
Figure 12_A0101_SEQ_0336
Figure 12_A0101_SEQ_0337
Figure 12_A0101_SEQ_0338
Figure 12_A0101_SEQ_0339
Figure 12_A0101_SEQ_0340
Figure 12_A0101_SEQ_0341
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Figure 12_A0101_SEQ_0345
Figure 12_A0101_SEQ_0346
Figure 12_A0101_SEQ_0347
Figure 12_A0101_SEQ_0348
Figure 12_A0101_SEQ_0349
Figure 12_A0101_SEQ_0350
Figure 12_A0101_SEQ_0351
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Figure 12_A0101_SEQ_0356
Figure 12_A0101_SEQ_0357
Figure 12_A0101_SEQ_0358
Figure 12_A0101_SEQ_0359
Figure 12_A0101_SEQ_0360
Figure 12_A0101_SEQ_0361
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Figure 12_A0101_SEQ_0364
Figure 12_A0101_SEQ_0365
Figure 12_A0101_SEQ_0366
Figure 12_A0101_SEQ_0367
Figure 12_A0101_SEQ_0368
Figure 12_A0101_SEQ_0369
Figure 12_A0101_SEQ_0370
Figure 12_A0101_SEQ_0371
Figure 12_A0101_SEQ_0372
Figure 12_A0101_SEQ_0373
Figure 12_A0101_SEQ_0374
Figure 12_A0101_SEQ_0375
Figure 12_A0101_SEQ_0376
Figure 12_A0101_SEQ_0377
Figure 12_A0101_SEQ_0378
Figure 12_A0101_SEQ_0379
Figure 12_A0101_SEQ_0380
Figure 12_A0101_SEQ_0381
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Figure 12_A0101_SEQ_0383
Figure 12_A0101_SEQ_0384
Figure 12_A0101_SEQ_0385
Figure 12_A0101_SEQ_0386
Figure 12_A0101_SEQ_0387
Figure 12_A0101_SEQ_0388
Figure 12_A0101_SEQ_0389
Figure 12_A0101_SEQ_0390
Figure 12_A0101_SEQ_0391
Figure 12_A0101_SEQ_0392
Figure 12_A0101_SEQ_0393
Figure 12_A0101_SEQ_0394
Figure 12_A0101_SEQ_0395
Figure 12_A0101_SEQ_0396
Figure 12_A0101_SEQ_0397
Figure 12_A0101_SEQ_0398
Figure 12_A0101_SEQ_0399
Figure 12_A0101_SEQ_0400
Figure 12_A0101_SEQ_0401
Figure 12_A0101_SEQ_0402
Figure 12_A0101_SEQ_0403
Figure 12_A0101_SEQ_0404
Figure 12_A0101_SEQ_0405
Figure 12_A0101_SEQ_0406
Figure 12_A0101_SEQ_0407
Figure 12_A0101_SEQ_0408
Figure 12_A0101_SEQ_0409
Figure 12_A0101_SEQ_0410
Figure 12_A0101_SEQ_0411
Figure 12_A0101_SEQ_0412
Figure 12_A0101_SEQ_0413
Figure 12_A0101_SEQ_0414
Figure 12_A0101_SEQ_0415
Figure 12_A0101_SEQ_0416
Figure 12_A0101_SEQ_0417
Figure 12_A0101_SEQ_0418
Figure 12_A0101_SEQ_0419
Figure 12_A0101_SEQ_0420
Figure 12_A0101_SEQ_0421
Figure 12_A0101_SEQ_0422
Figure 12_A0101_SEQ_0423
Figure 12_A0101_SEQ_0424
Figure 12_A0101_SEQ_0425
Figure 12_A0101_SEQ_0426
Figure 12_A0101_SEQ_0427
Figure 12_A0101_SEQ_0428
Figure 12_A0101_SEQ_0429
Figure 12_A0101_SEQ_0430
Figure 12_A0101_SEQ_0431
Figure 12_A0101_SEQ_0432
Figure 12_A0101_SEQ_0433
Figure 12_A0101_SEQ_0434
Figure 12_A0101_SEQ_0435
Figure 12_A0101_SEQ_0436
Figure 12_A0101_SEQ_0437
Figure 12_A0101_SEQ_0438
Figure 12_A0101_SEQ_0439
Figure 12_A0101_SEQ_0440
Figure 12_A0101_SEQ_0441
Figure 12_A0101_SEQ_0442
Figure 12_A0101_SEQ_0443
Figure 12_A0101_SEQ_0444
Figure 12_A0101_SEQ_0445
Figure 12_A0101_SEQ_0446
Figure 12_A0101_SEQ_0447
Figure 12_A0101_SEQ_0448
Figure 12_A0101_SEQ_0449
Figure 12_A0101_SEQ_0450
Figure 12_A0101_SEQ_0451
Figure 12_A0101_SEQ_0452
Figure 12_A0101_SEQ_0453
Figure 12_A0101_SEQ_0454
Figure 12_A0101_SEQ_0455
Figure 12_A0101_SEQ_0456
Figure 12_A0101_SEQ_0457
Figure 12_A0101_SEQ_0458
Figure 12_A0101_SEQ_0459
Figure 12_A0101_SEQ_0460
Figure 12_A0101_SEQ_0461
Figure 12_A0101_SEQ_0462
Figure 12_A0101_SEQ_0463
Figure 12_A0101_SEQ_0464
Figure 12_A0101_SEQ_0465
Figure 12_A0101_SEQ_0466
Figure 12_A0101_SEQ_0467
Figure 12_A0101_SEQ_0468
Figure 12_A0101_SEQ_0469
Figure 12_A0101_SEQ_0470
Figure 12_A0101_SEQ_0471
Figure 12_A0101_SEQ_0472
Figure 12_A0101_SEQ_0473
Figure 12_A0101_SEQ_0474
Figure 12_A0101_SEQ_0475
Figure 12_A0101_SEQ_0476
Figure 12_A0101_SEQ_0477
Figure 12_A0101_SEQ_0478
Figure 12_A0101_SEQ_0479
Figure 12_A0101_SEQ_0480
Figure 12_A0101_SEQ_0481
Figure 12_A0101_SEQ_0482
Figure 12_A0101_SEQ_0483
Figure 12_A0101_SEQ_0484
Figure 12_A0101_SEQ_0485
Figure 12_A0101_SEQ_0486
Figure 12_A0101_SEQ_0487
Figure 12_A0101_SEQ_0488
Figure 12_A0101_SEQ_0489
Figure 12_A0101_SEQ_0490
Figure 12_A0101_SEQ_0491
Figure 12_A0101_SEQ_0492
Figure 12_A0101_SEQ_0493
Figure 12_A0101_SEQ_0494
Figure 12_A0101_SEQ_0495
Figure 12_A0101_SEQ_0496
Figure 12_A0101_SEQ_0497
Figure 12_A0101_SEQ_0498
Figure 12_A0101_SEQ_0499
Figure 12_A0101_SEQ_0500
Figure 12_A0101_SEQ_0501
Figure 12_A0101_SEQ_0502
Figure 12_A0101_SEQ_0503
Figure 12_A0101_SEQ_0504
Figure 12_A0101_SEQ_0505
Figure 12_A0101_SEQ_0506
Figure 12_A0101_SEQ_0507
Figure 12_A0101_SEQ_0508
Figure 12_A0101_SEQ_0509
Figure 12_A0101_SEQ_0510
Figure 12_A0101_SEQ_0511
Figure 12_A0101_SEQ_0512
Figure 12_A0101_SEQ_0513
Figure 12_A0101_SEQ_0514
Figure 12_A0101_SEQ_0515
Figure 12_A0101_SEQ_0516
Figure 12_A0101_SEQ_0517
Figure 12_A0101_SEQ_0518
Figure 12_A0101_SEQ_0519
Figure 12_A0101_SEQ_0520
Figure 12_A0101_SEQ_0521
Figure 12_A0101_SEQ_0522
Figure 12_A0101_SEQ_0523
Figure 12_A0101_SEQ_0524
Figure 12_A0101_SEQ_0525
Figure 12_A0101_SEQ_0526
Figure 12_A0101_SEQ_0527
Figure 12_A0101_SEQ_0528
Figure 12_A0101_SEQ_0529
Figure 12_A0101_SEQ_0530
Figure 12_A0101_SEQ_0531
Figure 12_A0101_SEQ_0532
Figure 12_A0101_SEQ_0533
Figure 12_A0101_SEQ_0534
Figure 12_A0101_SEQ_0535
Figure 12_A0101_SEQ_0536
Figure 12_A0101_SEQ_0537
Figure 12_A0101_SEQ_0538
Figure 12_A0101_SEQ_0539
Figure 12_A0101_SEQ_0540
Figure 12_A0101_SEQ_0541
Figure 12_A0101_SEQ_0542
Figure 12_A0101_SEQ_0543
Figure 12_A0101_SEQ_0544
Figure 12_A0101_SEQ_0545
Figure 12_A0101_SEQ_0546
Figure 12_A0101_SEQ_0547
Figure 12_A0101_SEQ_0548
Figure 12_A0101_SEQ_0549
Figure 12_A0101_SEQ_0550
Figure 12_A0101_SEQ_0551
Figure 12_A0101_SEQ_0552
Figure 12_A0101_SEQ_0553
Figure 12_A0101_SEQ_0554
Figure 12_A0101_SEQ_0555
Figure 12_A0101_SEQ_0556
Figure 12_A0101_SEQ_0557
Figure 12_A0101_SEQ_0558
Figure 12_A0101_SEQ_0559
Figure 12_A0101_SEQ_0560
Figure 12_A0101_SEQ_0561
Figure 12_A0101_SEQ_0562
Figure 12_A0101_SEQ_0563
Figure 12_A0101_SEQ_0564
Figure 12_A0101_SEQ_0565
Figure 12_A0101_SEQ_0566
Figure 12_A0101_SEQ_0567
Figure 12_A0101_SEQ_0568
Figure 12_A0101_SEQ_0569
Figure 12_A0101_SEQ_0570
Figure 12_A0101_SEQ_0571
Figure 12_A0101_SEQ_0572
Figure 12_A0101_SEQ_0573
Figure 12_A0101_SEQ_0574
Figure 12_A0101_SEQ_0575
Figure 12_A0101_SEQ_0576
Figure 12_A0101_SEQ_0577
Figure 12_A0101_SEQ_0578
Figure 12_A0101_SEQ_0579
Figure 12_A0101_SEQ_0580
Figure 12_A0101_SEQ_0581
Figure 12_A0101_SEQ_0582
Figure 12_A0101_SEQ_0583
Figure 12_A0101_SEQ_0584
Figure 12_A0101_SEQ_0585
Figure 12_A0101_SEQ_0586
Figure 12_A0101_SEQ_0587
Figure 12_A0101_SEQ_0588
Figure 12_A0101_SEQ_0589
Figure 12_A0101_SEQ_0590
Figure 12_A0101_SEQ_0591
Figure 12_A0101_SEQ_0592
Figure 12_A0101_SEQ_0593
Figure 12_A0101_SEQ_0594
Figure 12_A0101_SEQ_0595
Figure 12_A0101_SEQ_0596
Figure 12_A0101_SEQ_0597
Figure 12_A0101_SEQ_0598
Figure 12_A0101_SEQ_0599
Figure 12_A0101_SEQ_0600
Figure 12_A0101_SEQ_0601
Figure 12_A0101_SEQ_0602
Figure 12_A0101_SEQ_0603
Figure 12_A0101_SEQ_0604
Figure 12_A0101_SEQ_0605
Figure 12_A0101_SEQ_0606
Figure 12_A0101_SEQ_0607
Figure 12_A0101_SEQ_0608
Figure 12_A0101_SEQ_0609
Figure 12_A0101_SEQ_0610
Figure 12_A0101_SEQ_0611
Figure 12_A0101_SEQ_0612
Figure 12_A0101_SEQ_0613
Figure 12_A0101_SEQ_0614
Figure 12_A0101_SEQ_0615
Figure 12_A0101_SEQ_0616
Figure 12_A0101_SEQ_0617
Figure 12_A0101_SEQ_0618
Figure 12_A0101_SEQ_0619
Figure 12_A0101_SEQ_0620
Figure 12_A0101_SEQ_0621
Figure 12_A0101_SEQ_0622
Figure 12_A0101_SEQ_0623
Figure 12_A0101_SEQ_0624
Figure 12_A0101_SEQ_0625
Figure 12_A0101_SEQ_0626
Figure 12_A0101_SEQ_0627
Figure 12_A0101_SEQ_0628
Figure 12_A0101_SEQ_0629
Figure 12_A0101_SEQ_0630
Figure 12_A0101_SEQ_0631
Figure 12_A0101_SEQ_0632
Figure 12_A0101_SEQ_0633
Figure 12_A0101_SEQ_0634
Figure 12_A0101_SEQ_0635
Figure 12_A0101_SEQ_0636
Figure 12_A0101_SEQ_0637
Figure 12_A0101_SEQ_0638
Figure 12_A0101_SEQ_0639
Figure 12_A0101_SEQ_0640
Figure 12_A0101_SEQ_0641
Figure 12_A0101_SEQ_0642
Figure 12_A0101_SEQ_0643
Figure 12_A0101_SEQ_0644
Figure 12_A0101_SEQ_0645
Figure 12_A0101_SEQ_0646
Figure 12_A0101_SEQ_0647
Figure 12_A0101_SEQ_0648
Figure 12_A0101_SEQ_0649
Figure 12_A0101_SEQ_0650
Figure 12_A0101_SEQ_0651
Figure 12_A0101_SEQ_0652
Figure 12_A0101_SEQ_0653
Figure 12_A0101_SEQ_0654
Figure 12_A0101_SEQ_0655
Figure 12_A0101_SEQ_0656
Figure 12_A0101_SEQ_0657
Figure 12_A0101_SEQ_0658
Figure 12_A0101_SEQ_0659
Figure 12_A0101_SEQ_0660
Figure 12_A0101_SEQ_0661
Figure 12_A0101_SEQ_0662
Figure 12_A0101_SEQ_0663
Figure 12_A0101_SEQ_0664
Figure 12_A0101_SEQ_0665
Figure 12_A0101_SEQ_0666
Figure 12_A0101_SEQ_0667
Figure 12_A0101_SEQ_0668
Figure 12_A0101_SEQ_0669
Figure 12_A0101_SEQ_0670
Figure 12_A0101_SEQ_0671
Figure 12_A0101_SEQ_0672
Figure 12_A0101_SEQ_0673
Figure 12_A0101_SEQ_0674
Figure 12_A0101_SEQ_0675
Figure 12_A0101_SEQ_0676
Figure 12_A0101_SEQ_0677
Figure 12_A0101_SEQ_0678
Figure 12_A0101_SEQ_0679
Figure 12_A0101_SEQ_0680
Figure 12_A0101_SEQ_0681
Figure 12_A0101_SEQ_0682
Figure 12_A0101_SEQ_0683
Figure 12_A0101_SEQ_0684
Figure 12_A0101_SEQ_0685
Figure 12_A0101_SEQ_0686
Figure 12_A0101_SEQ_0687
Figure 12_A0101_SEQ_0688
Figure 12_A0101_SEQ_0689
Figure 12_A0101_SEQ_0690
Figure 12_A0101_SEQ_0691
Figure 12_A0101_SEQ_0692
Figure 12_A0101_SEQ_0693
Figure 12_A0101_SEQ_0694
Figure 12_A0101_SEQ_0695
Figure 12_A0101_SEQ_0696
Figure 12_A0101_SEQ_0697
Figure 12_A0101_SEQ_0698
Figure 12_A0101_SEQ_0699
Figure 12_A0101_SEQ_0700
Figure 12_A0101_SEQ_0701
Figure 12_A0101_SEQ_0702
Figure 12_A0101_SEQ_0703
Figure 12_A0101_SEQ_0704
Figure 12_A0101_SEQ_0705
Figure 12_A0101_SEQ_0706
Figure 12_A0101_SEQ_0707
Figure 12_A0101_SEQ_0708
Figure 12_A0101_SEQ_0709
Figure 12_A0101_SEQ_0710
Figure 12_A0101_SEQ_0711
Figure 12_A0101_SEQ_0712
Figure 12_A0101_SEQ_0713
Figure 12_A0101_SEQ_0714
Figure 12_A0101_SEQ_0715
Figure 12_A0101_SEQ_0716
Figure 12_A0101_SEQ_0717
Figure 12_A0101_SEQ_0718
Figure 12_A0101_SEQ_0719
Figure 12_A0101_SEQ_0720
Figure 12_A0101_SEQ_0721
Figure 12_A0101_SEQ_0722
Figure 12_A0101_SEQ_0723
Figure 12_A0101_SEQ_0724
Figure 12_A0101_SEQ_0725
Figure 12_A0101_SEQ_0726
Figure 12_A0101_SEQ_0727
Figure 12_A0101_SEQ_0728
Figure 12_A0101_SEQ_0729
Figure 12_A0101_SEQ_0730
Figure 12_A0101_SEQ_0731
Figure 12_A0101_SEQ_0732
Figure 12_A0101_SEQ_0733
Figure 12_A0101_SEQ_0734
Figure 12_A0101_SEQ_0735
Figure 12_A0101_SEQ_0736
Figure 12_A0101_SEQ_0737
Figure 12_A0101_SEQ_0738
Figure 12_A0101_SEQ_0739
Figure 12_A0101_SEQ_0740
Figure 12_A0101_SEQ_0741
Figure 12_A0101_SEQ_0742
Figure 12_A0101_SEQ_0743
Figure 12_A0101_SEQ_0744
Figure 12_A0101_SEQ_0745
Figure 12_A0101_SEQ_0746
Figure 12_A0101_SEQ_0747
Figure 12_A0101_SEQ_0748
Figure 12_A0101_SEQ_0749
Figure 12_A0101_SEQ_0750
Figure 12_A0101_SEQ_0751
Figure 12_A0101_SEQ_0752
Figure 12_A0101_SEQ_0753
Figure 12_A0101_SEQ_0754
Figure 12_A0101_SEQ_0755
Figure 12_A0101_SEQ_0756
Figure 12_A0101_SEQ_0757
Figure 12_A0101_SEQ_0758
Figure 12_A0101_SEQ_0759
Figure 12_A0101_SEQ_0760
Figure 12_A0101_SEQ_0761
Figure 12_A0101_SEQ_0762
Figure 12_A0101_SEQ_0763
Figure 12_A0101_SEQ_0764
Figure 12_A0101_SEQ_0765
Figure 12_A0101_SEQ_0766
Figure 12_A0101_SEQ_0767
Figure 12_A0101_SEQ_0768
Figure 12_A0101_SEQ_0769
Figure 12_A0101_SEQ_0770
Figure 12_A0101_SEQ_0771
Figure 12_A0101_SEQ_0772
Figure 12_A0101_SEQ_0773
Figure 12_A0101_SEQ_0774
Figure 12_A0101_SEQ_0775
Figure 12_A0101_SEQ_0776
Figure 12_A0101_SEQ_0777
Figure 12_A0101_SEQ_0778
Figure 12_A0101_SEQ_0779
Figure 12_A0101_SEQ_0780
Figure 12_A0101_SEQ_0781
Figure 12_A0101_SEQ_0782
Figure 12_A0101_SEQ_0783
Figure 12_A0101_SEQ_0784
Figure 12_A0101_SEQ_0785
Figure 12_A0101_SEQ_0786
Figure 12_A0101_SEQ_0787
Figure 12_A0101_SEQ_0788
Figure 12_A0101_SEQ_0789
Figure 12_A0101_SEQ_0790
Figure 12_A0101_SEQ_0791
Figure 12_A0101_SEQ_0792
Figure 12_A0101_SEQ_0793
Figure 12_A0101_SEQ_0794
Figure 12_A0101_SEQ_0795
Figure 12_A0101_SEQ_0796
Figure 12_A0101_SEQ_0797
Figure 12_A0101_SEQ_0798
Figure 12_A0101_SEQ_0799
Figure 12_A0101_SEQ_0800
Figure 12_A0101_SEQ_0801
Figure 12_A0101_SEQ_0802
Figure 12_A0101_SEQ_0803
Figure 12_A0101_SEQ_0804
Figure 12_A0101_SEQ_0805
Figure 12_A0101_SEQ_0806
Figure 12_A0101_SEQ_0807
Figure 12_A0101_SEQ_0808
Figure 12_A0101_SEQ_0809
Figure 12_A0101_SEQ_0810
Figure 12_A0101_SEQ_0811
Figure 12_A0101_SEQ_0812
Figure 12_A0101_SEQ_0813
Figure 12_A0101_SEQ_0814
Figure 12_A0101_SEQ_0815
Figure 12_A0101_SEQ_0816
Figure 12_A0101_SEQ_0817
Figure 12_A0101_SEQ_0818
Figure 12_A0101_SEQ_0819
Figure 12_A0101_SEQ_0820
Figure 12_A0101_SEQ_0821
Figure 12_A0101_SEQ_0822
Figure 12_A0101_SEQ_0823
Figure 12_A0101_SEQ_0824
Figure 12_A0101_SEQ_0825
Figure 12_A0101_SEQ_0826
Figure 12_A0101_SEQ_0827
Figure 12_A0101_SEQ_0828
Figure 12_A0101_SEQ_0829
Figure 12_A0101_SEQ_0830
Figure 12_A0101_SEQ_0831
Figure 12_A0101_SEQ_0832
Figure 12_A0101_SEQ_0833
Figure 12_A0101_SEQ_0834
Figure 12_A0101_SEQ_0835
Figure 12_A0101_SEQ_0836
Figure 12_A0101_SEQ_0837
Figure 12_A0101_SEQ_0838
Figure 12_A0101_SEQ_0839
Figure 12_A0101_SEQ_0840
Figure 12_A0101_SEQ_0841
Figure 12_A0101_SEQ_0842
Figure 12_A0101_SEQ_0843
Figure 12_A0101_SEQ_0844
Figure 12_A0101_SEQ_0845
Figure 12_A0101_SEQ_0846
Figure 12_A0101_SEQ_0847
Figure 12_A0101_SEQ_0848
Figure 12_A0101_SEQ_0849
Figure 12_A0101_SEQ_0850
Figure 12_A0101_SEQ_0851
Figure 12_A0101_SEQ_0852
Figure 12_A0101_SEQ_0853
Figure 12_A0101_SEQ_0854
Figure 12_A0101_SEQ_0855
Figure 12_A0101_SEQ_0856
Figure 12_A0101_SEQ_0857
Figure 12_A0101_SEQ_0858
Figure 12_A0101_SEQ_0859
Figure 12_A0101_SEQ_0860
Figure 12_A0101_SEQ_0861
Figure 12_A0101_SEQ_0862
Figure 12_A0101_SEQ_0863
Figure 12_A0101_SEQ_0864
Figure 12_A0101_SEQ_0865
Figure 12_A0101_SEQ_0866
Figure 12_A0101_SEQ_0867
Figure 12_A0101_SEQ_0868
Figure 12_A0101_SEQ_0869
Figure 12_A0101_SEQ_0870
Figure 12_A0101_SEQ_0871
Figure 12_A0101_SEQ_0872
Figure 12_A0101_SEQ_0873
Figure 12_A0101_SEQ_0874
Figure 12_A0101_SEQ_0875
Figure 12_A0101_SEQ_0876
Figure 12_A0101_SEQ_0877
Figure 12_A0101_SEQ_0878
Figure 12_A0101_SEQ_0879
Figure 12_A0101_SEQ_0880
Figure 12_A0101_SEQ_0881
Figure 12_A0101_SEQ_0882
Figure 12_A0101_SEQ_0883
Figure 12_A0101_SEQ_0884
Figure 12_A0101_SEQ_0885
Figure 12_A0101_SEQ_0886
Figure 12_A0101_SEQ_0887
Figure 12_A0101_SEQ_0888
Figure 12_A0101_SEQ_0889
Figure 12_A0101_SEQ_0890
Figure 12_A0101_SEQ_0891
Figure 12_A0101_SEQ_0892
Figure 12_A0101_SEQ_0893
Figure 12_A0101_SEQ_0894
Figure 12_A0101_SEQ_0895

Claims (20)

  1. 一種PH20變異體或其片段，其在包含SEQ ID NO: 3之胺基酸序列的變異體中之至少一位置包含胺基酸殘基取代、刪除或插入，且其具有較野生型PH20的聚集溫度更高的聚集溫度。
  2. 如請求項1所述之PH20變異體或其片段，其中該PH20變異體或其片段在具有SEQ ID NO: 3之該胺基酸序列的該變異體中之自由R39、D65至L68、N82、T84、I102至I105、T132至Y134、N166、L179至T182、T185至K187、V241至K244、N266至Q269、P271、V272、K290至P292、Q311至K314、G340至N363、L441、S442、D451至D453、D461、V463及D461至V463組成之群組選擇之至少一位置包含該胺基酸殘基取代。
  3. 如請求項2所述之PH20變異體或其片段，其中該胺基酸殘基取代包含自由R39K、D65A、E66A、P67A、L68A、N82A、T84N、I102A、D103A、S104A、S104N、I105A、I105Q、T132A、T132S、F133A、Y134A、N166A、N166K、L179A、L179S、L179I、L179F、S180T、S180A、L181A、L181M、T182A、T185A、E186A、E186D、K187A、V241A、E242A、I243A、K244A、N266A、T267A、Q268A、Q268D、Q268I、Q268N、Q269A、P271A、V272A、K290A、I291A、I291G、I291L、P292A、P292D、Q311A、V312A、L313A、L313P、L313M、K314A、G340Q、S341H、S341D、S341T、W342I、W342D、W342H、W342L、E343V、E343S、E343Y、E343Q、N344F、N344I、T345E、T345K、T345S、R346M、R346F、R346L、R346T、R346S、R346A、T347Q、T347E、T347V、T347W、T347H、T347S、K348Q、K348F、K348D、K348T、K348E、K348M、E349L、E349W、E349A、S350Q、S350I、S350D、S350T、S350E、S350N、Q352E、Q352G、Q352Y、Q352W、Q352T、A353E、A353Y、A353H、A353K、I354E、I354Q、I354S、I354V、I354A、I354N、I354T、I354R、I354W、I354L、K355Q、K355H、K355D、E356M、E356F、E356I、E356L、E356Q、E356V、E356D、 Y357W、Y357F、M358V、M358R、M358Y、M358L、D359K、D359V、D359Y、D359Q、D359T、D359S、D359E、T360Y、T360R、T360L、T360D、T360S、T361M、T361E、T361H、T361L、T361D、T361I、L362A、N363M、N363E、L441A、S442A、D451A、D451S、T452A、T452D、T452H、T452K、T452G、T452P、T452M、T452F、D453A、D461R、D461A、G462A、V463Y及V463A組成之群組選擇之至少一者。
  4. 如請求項1至3之任一項所述之PH20變異體或其片段，在C端及/或N端更包含至少一胺基酸殘基的刪除。
  5. 如請求項4所述之PH20變異體或其片段，其中該至少一胺基酸殘基係藉由在自由M1至P42組成之群組選擇之一胺基酸殘基之前自N端切割而刪除。
  6. 如請求項5所述之PH20變異體或其片段，其中該至少一胺基酸殘基係藉由在L36、N37、F38、R39、A40、P41或P42之一胺基酸殘基之前自N端切割而刪除。
  7. 如請求項4所述之PH20變異體或其片段，其中該至少一胺基酸殘基係藉由在自由V455至L509組成之群組選擇之一胺基酸殘基之後自C端切割而刪除。
  8. 如請求項7所述之PH20變異體或其片段，其中該至少一胺基酸殘基係藉由在自由V455至S490組成之群組選擇之一胺基酸殘基之後自C端切割而刪除。
  9. 如請求項7所述之PH20變異體或其片段，其中該至少一胺基酸殘基係藉由在V455、D456、C458、D461、C464、I465、D466、A467、F468、K470、P471、P472、M473、E474、T475、E476、P478、I480、Y482、A484、P486、T488或S490胺基酸殘基之後自C端切割而刪除
  10. 如請求項4所述之PH20變異體或其片段，其中該至少一胺基酸殘基係藉由在F38之胺基酸殘基之前自N端及在F468之胺基酸殘基之後自C端切割而刪除。
  11. 如請求項1～3及5～10之任一項所述之PH20變異體或其片段，其中該PH20變異體或其片段包含選自由SEQ ID NOs: 163至316之胺基酸序列組成之群組之胺基酸序列。
  12. 如請求項4所述之PH20變異體或其片段，其中該PH20變異體或其片段包含選自由SEQ ID NOs: 163至316之胺基酸序列組成之群組之胺基酸序列。
  13. 一種用於治療癌症之組成物，包含如請求項1至12之任一項所述之PH20變異體或其片段。
  14. 如請求項13所述之組成物，其中該組成物與一抗癌藥物用於合併療法。
  15. 如請求項14所述之組成物，其中該抗癌藥物為一免疫腫瘤試劑。
  16. 如請求項15所述之組成物，其中該免疫腫瘤試劑包含一免疫檢查點抑制劑。
  17. 一種核酸，編碼如請求項1至12之任一項所述之PH20變異體或其片段。
  18. 一種重組表現載體，包含如請求項17所述之核酸。
  19. 一種宿主細胞，以如請求項18所述之重組表現載體轉形。
  20. 一種製造PH20變異體或其片段的方法，包含培養如請求項19所述之宿主細胞。
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